cytotoxic and conjugated antibody drug peptides thereof

专利摘要:
cytotoxic peptides and antibody drug conjugates thereof. the present invention relates to cytotoxic pentapeptides, antibody drug conjugates thereof, and methods for using them to treat cancer.
公开号:BR112014012609A2
申请号:R112014012609-7
申请日:2012-11-07
公开日:2020-10-20
发明作者:Matthew David DOROSKI；Andreas Maderna；Christopher John O'Donnell；Chakarapani Subramanyam；Beth Cooper Vetelino；Russel George Dushin；Pavel Strop；Edmund Idris Graziani
申请人:Pfizer Inc；
IPC主号:

专利说明:

[001] [001] This claim claims the benefits of United States Provisional Order No. 61 / 561.255, filed on November 17, 2011, and United States Provisional Application No. 61 / 676,423, deposited on July 27, 2012, both of which are fully incorporated herein. FIELD OF THE INVENTION
[002] [002] The present invention relates to new compounds based on peptides useful as fillers in antibody drug conjugates (ADC's), and useful linker compounds in connection with ADC's. The present invention further relates to compositions including the aforementioned fillers, filler binders and ADC’s, and methods for using these fillers, filler binders and ADC’s, to treat pathological conditions including cancer. BACKGROUND
[003] [003] The conjugation of drugs to antibodies, either directly or through ligands, involves a consideration of a variety of factors, including the identity and location of the chemical group for the conjugation of the drug, the mechanism of drug release, the structural elements that provide the drug release, and the structural modification for the released free drug. In addition, if the drug is to be released after internalization of the antibody, the mechanism of drug release must correspond to the intracellular traffic of the conjugate.
[004] [004] Although a variety of different classes of drugs have been tested for distribution by antibodies, only a few classes of drugs have proven effective as antibody drug conjugates, while having an adequate toxicity profile.
[005] [005] One class is auristatins, derived from the natural product dolastatin 10.
[006] [006] The present invention relates to cytotoxic pentapeptides and antibody drug conjugates thereof represented by the formula:
[007] [007] or a pharmaceutically acceptable salt or solvate thereof, where, independently for each occurrence, 1-2 R3A
[008] [008] R1 is hydrogen, C1-C8 alkyl, or C1-C8 haloalkyl, or R1 is a linker or a linker-antibody, such as Y O Z Z
[009] [009] Y is -C2-C20 alkylene-, -C2-C20 heteroalkylene-, C3-C8 carbocyclo-, -arylene-, -C3-C8 heterocyclo-, -Cl-C10alkylene-arylene-, -arylene-Cl-Cl0alkylene- , -Cl- Cl0 alkylene- (C3-
[010] [010] C8carbocycle) -, - (C3-C8carbocycle) -Cl-C10alkylene-, -Cl-Cl0alkylene- (C3-C8 heterocycle) - or - (C3-C8 heterocycle) -Cl-Cl0alkylene-; Z is,,,,, H H N N H N O L N O O O, O O, O H The L N H N O
[011] [011] G is halogen, -OH, -SH or -S-C1-C6 alkyl;
[012] [012] L is an antibody;
[013] [013] R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl;
[014] [014] R3A and R3B are defined as one of the following:
[015] [015] R3A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and
[016] [016] R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; or
[017] [017] R3A and R3B taken together are C2-C8 alkylene or C1-C8 heteroalkylene;
[018] [018] R4A and R4B are defined as one of the following:
[019] [019] R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and
[020] [020] R4B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or
[021] [021] R4A and R4B taken together are C2-C8 alkylene or C1-C8 heteroalkylene;
[022] [022] R5 is,,,,,,,,,,,,, C1-C10 heterocyclyl, C3-C8 carbocyclyl and C6-C14 aryl optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from the group consisting of -C1-C8 alkyl, -C1-C8 alkyl-N (R ') 2, -C1-C8 alkyl-C (O) R', -C1-C8 alkyl-C (O) OR '-O- (C1-C8 alkyl), -C (O) R ', -OC (O) R', -C (O) OR ', -C (O) N (R') 2, -NHC (O) R ' , -S (O) 2R ', -S (O) R', -OH, halogen, -N3, -N (R ') 2, -CN, -NHC (= NH) NH2, -NHCONH2, -S ( = O) 2R 'and -SR', where each R 'is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl, or two R' can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; O
[023] [023] or R5 is, R13, or
[024] [024] R6 is hydrogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -C1-C8 haloalkyl;
[025] [025] R12 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl;
[026] [026] R13 is C1-C10 heterocyclyl; and
[027] [027] R7 is F, Cl, I, Br, NO2, CN and CF3;
[028] [028] h is 1, 2, 3, 4 or 5; and
[029] [029] X is O or S;
[030] [030] since when R3A is hydrogen, X is S.
[031] [031] The present invention relates to cytotoxic pentapeptides and antibody drug conjugates represented by the formula:
[032] [032] or a pharmaceutically acceptable salt or solvate thereof, where, independently for each occurrence, 1-2 R3A
[033] [033] R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl;
[034] [034] R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl;
[035] [035] R3A and R3B are defined as one of the following:
[036] [036] R3A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and
[037] [037] R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl, halogen or hydrogen; or
[038] [038] R3A and R3B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
[039] [039] R4A and R4B are defined as one of the following:
[040] [040] R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and
[041] [041] R4B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or
[042] [042] R4A and R4B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
[043] [043] R5 is
[044] [044] optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -O- (C1-C8 alkyl), -C (O) R ', -OC (O ) R ', -C (O) OR', -C (O) NH2, -C (O) NHR ', -C (O) N (R') 2, -NHC (O) R ', -S ( O) 2R ', -S (O) R', -OH, halogen, -N3, -NH2, -NH (R '), -N (R') 2, -CN, -NHC (= NH) NH2, -NHCONH2, -S (= O) 2R 'and -SR', where each R 'is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl;
[045] [045] R11 is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, or R11 is a linker or linker-antibody, such as Y O Z Z
[046] [046] Y is C2-C20 alkylene or C2-C20 heteroalkylene; C3-C8 carbocyclo-, -arylene-, - C3-C8heterocyclo-, -Cl-C10alkylene-arylene-, -arylene-Cl-Cl0Clalkylene-, -Cl-Cl0alkylene- (C3-
[047] [047] C8carbocycle) -, - (C3-C8carbocycle) -Cl-C10alkylene-, -Cl-C10 alkylene- (C3-C8 heterocycle) -, or - (C3-C8 heterocycle) -Cl-Cl0alkylene-; Z is,,,,, H H N N H N O L N O O O, O O, O H The L N H N O
[048] [048] G is halogen, -OH, -SH or -S-C1-C6 alkyl;
[049] [049] L is an antibody;
[050] [050] R7 is F, Cl, I, Br, NO2, CN and CF3;
[051] [051] h is 1, 2, 3, 4 or 5; and
[052] [052] X is O or S.
[053] [053] Another aspect of the invention relates to pharmaceutical compositions including an effective amount of any of the above-mentioned compounds and / or any of the above-mentioned antibody drug conjugates and a pharmaceutically acceptable carrier or carrier.
[054] [054] Another aspect of the invention relates to a method of using an effective amount of any of the above-mentioned compounds and / or any of the above-mentioned antibody drug conjugates to treat cancer by administering it to a patient in need of even an effective amount of said compound and / or conjugate.
[055] [055] Another aspect of the invention relates to a cancer treatment method in which said cancer includes a tumor, metastasis, or other disease or disorder characterized by uncontrolled cell growth, in which said cancer is selected from the group consisting of in carcinomas of the bladder, breast, cervix, colon, gliomas, endometrium, kidney, lung, esophagus, ovary, prostate, pancreas, melanoma, stomach and testicles. BRIEF DESCRIPTION OF THE FIGURES
[056] [056] Figure 1 represents a graph of anti-tumor activity of four conjugates (each administered at 1 mg / kg, Q4dx4) plotted as a tumor column over time.
[057] [057] Figure 2 represents a graph of anti-tumor activity of six conjugates (each administered at 1 mg / kg, Q4dx4) plotted as drug-treated tumor volume / vehicle-treated tumor volume over time.
[058] [058] Figure 3 represents the results of the H (C) - # D54 and H (C) -vcMMAE test at 1 mg / kg s
[059] [059] Figures 4A, 4B and 4C represent [A] H (C) - # D54 test results and
[060] [060] Figures 5A, 5B, 5C, 5D, 5E and 5F represent the dose response results of the [A] H (C) - # D54, [B] H (C) -vcMMAE, [C] H (C) -mcMMAF and [D] H (K) - MCC-DM1 in an in vivo model of N87 mouse xenograft; [E] a comparison of H (C) - # D54 and H (C) -vcMMAE; and [F] a T / C comparison for all four conjugates. The mice were treated q4dx4, starting on day 1.
[061] [061] Figure 6 represents the dose response results of the H (C) - # A115 test at 1 mpk, 3 mpk and 10 mpk, in an in vivo model of N87 mouse xenograft.
[062] [062] Figure 7 presents data comparing humanized antibody hu08 conjugated to vc-0101 or mc-3377, tested in an in vivo xenograft model with PC3MM2 cells, a human prostate cancer cell line that expresses the IL-13R 2 receptor.
[063] [063] Figures 8A to E show [A] the efficacy of mouse-human chimeric anti-Notch ADCs dosed at 5mg / kg in HCC2429 lung xenografts; [B and C] the effectiveness of rat-human chimeric anti-Notch ADCs dosed at 5mg / kg in MDA-MB-468 breast xenografts; [D and E] the efficacy of rat-human chimeric anti-Notch ADCs dosed at 5mg / kg in N87 gastric xenograft. DETAILED DESCRIPTION
[064] [064] The present invention relates to cytotoxic pentapeptides, antibody drug conjugates comprising said cytotoxic pentapeptides, and methods for using them to treat cancer and other pathological conditions. The invention also relates to methods of using such compounds and / or conjugates in vitro, in situ, and in vivo for the detection, diagnosis or treatment of mammalian cells, or associated pathological conditions.
[065] [065] Unless otherwise indicated, the following terms and phrases as used here are intended to have the following meanings. When trade names are used here, the trade name includes the product formulation, the generic drug, and the active pharmaceutical ingredient (s) of the trade name product, unless otherwise stated by context.
[066] [066] The term "antibody" (or "Ab") here is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (for example, bispecific antibodies), and antibody fragments that exhibit the desired biological activity. An intact antibody primarily has two regions: a variable region and a constant region. The variable region binds to and interacts with a target antigen. The variable region includes a determinant complementarity region (CDR) that recognizes and binds to a specific binding site on a particular antigen. The constant region can be recognized by and interact with the immune system (see, for example, Janeway et al., 2001, Immuno. Biology, 5th Ed., Garland Publishing, New York). An antibody can be of any type or class (for example, IgG, IgE, IgM, IgD, and IgA) or subclass (for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). The antibody can be derived from any suitable species. In some embodiments, the antibody is of human or murine origin. An antibody can be, for example, human, humanized or chimeric.
[067] [067] The terms "specifically bind" and "specific binding" refer to agglutination of antibody to a predetermined antigen. Typically, the antibody binds with an affinity of at least about 1x107 M-1, and binds to the predetermined antigen with an affinity that is at least twice as high as its affinity for agglutination to a non-specific antigen (for example, BSA, casein) other than the predetermined antigen or a closely related antigen.
[068] [068] The term "monoclonal antibody", as used here, refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in small quantities. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The "monoclonal" modifier indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any particular method.
[069] [069] The term "monoclonal antibodies" specifically includes "chimeric" antibodies in which a portion of the light and / or heavy chain is identical to or homologous with the corresponding sequence of antibodies derived from a particular species or belonging to a class or sub-class of particular antibody, while the rest of the chain (s) is identical to or homologous to the corresponding antibody sequences derived from other species or belonging to another class or subclass of antibody, as well as fragments of such antibodies, as long as they have the desired biological activity.
[070] [070] As used here, "H (C) -" refers to trastuzumab (trade name HERCEPTIN®) which is a monoclonal antibody that interferes with the HER2 / neu receptor, linked through one of its cystines to the compound of the invention. As used herein, "H (K) -" refers to trastuzumab which is a monoclonal antibody that interferes with the HER2 / neu receptor, linked via one of its lysines to the compound of the invention.
[071] [071] An "intact antibody" is one that comprises an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2, CH3 and CH4, as appropriate for the class of antibody. The constant domains can be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.
[072] [072] An intact antibody can have one or more "effector functions", which refer to those biological activities attributable to the Fc region (for example, a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis.
[073] [073] An "antibody fragment" comprises a portion of an intact antibody, preferably comprising the variable or antigen agglutination region thereof. Examples of antibody fragments include Fab, Fab ', F (ab') 2, and Fv fragments, diabodies, tribodies, tetribodies, linear antibodies, single chain antibody molecules, scFv, scFv-Fc, multispecific antibody fragments formed from antibody fragment (s), a fragment (s) produced by a Fab expression library, or epitope agglutination fragments from any of the above that specifically binds to a target antigen (e.g., a cancer cell antigen, a viral antigen or a microbial antigen).
[074] [074] The term "variable" in the context of an antibody refers to certain portions of the variable domains of the antibody that differ extensively in sequence and are used in the agglutination and specificity of each particular antibody to its particular antigen. This variability is concentrated in three segments called "hypervariable regions" in the light and heavy chain variable domains. The most highly conserved portions of variable domains are called frame regions (FRs). The variable domains of native light and heavy chains each comprise four FRs connected by three hypervariable regions.
[075] [075] The term "hypervariable region", when used here, refers to the amino acid residues of an antibody that are responsible for agglutination to antigen. The hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (for example, residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the chain variable domain mild and 31-35 (H1), 50-65 (H2) and 95-102 (L3) in the heavy chain variable domain; Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or those residues from a "hypervariable cycle" (for example, residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the variable domain light chain and 26-32 (H1), 53-55 (142) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-917). RF residues are those residues from the variable domain other than residues from the hypervariable region as defined herein.
[076] [076] An "Fv single chain" or "scFv" antibody fragment comprises the V.sub.H and V.sub.L domains of an antibody, where these domains are present in a single polypeptide chain. Typically, the Fv polypeptide still comprises a polypeptide linker between the V.sub.H and V.sub.L domains that allows scFv to form the desired structure for antigen agglutination. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.
[077] [077] The term "diabody" refers to small antibody fragments with two antigen agglutination sites, whose fragments comprise a variable heavy domain (VH) connected to a variable light domain (VL) on the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain and create two antigen-binding sites. Diabodies are more fully described in, for example, EP 0 404 097; WO 93/11161; and Hollinger et al., 1993, Proc. Natl. Acad.
[078] [078] "Humanized" forms of non-human antibodies (eg, rodents) are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (receptor antibody), in which residues from a hypervariable region of the receptor are replaced by residues from a hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and ability. In some cases, residues from the human immunoglobulin frame region (FR) are replaced by corresponding non-human residues. In addition, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine the performance of the antibody. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, where all or substantially all of the hypervariable cycles correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those in a sequence of human immunoglobulin. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., 1986, Nature 321: 522-525; Riechmann et al., 1988, Nature 332: 323-
[079] [079] As used herein, "isolated" means separated from other components of (a) a natural source, such as a cell culture or plant or animal cell, or (b) a synthetic organic chemical reaction mixture. As used herein, "purified" means that, when isolated, the isolate contains at least 95%, and in another aspect at least 98%, of a compound (e.g., a conjugate) by weight of the isolate.
[080] [080] An "isolated" antibody is one that has been identified and separated and / or recovered from a component in its natural environment. Contaminating components of its natural environment are materials that can interfere with diagnostic or therapeutic uses for the antibody, and can include enzymes, hormones, and other protein or non-protein solutes. In preferred embodiments, the antibody will be purified (1) to more than 95% by weight of the antibody as determined by the Lowry method, and more preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by the use of a rotating cup sequencer, or (3) for homogeneity by SDS-PAGE under conditions of reduction or non-reduction using Coomassie blue or, preferably, silver dye. Isolated antibody includes the antibody in situ within recombinant cells as long as at least one component of the antibody's natural environment is not present. Generally, however, the isolated antibody will be prepared by at least one purification step.
[081] [081] An antibody that "induces apoptosis" is one that induces programmed cell death as determined by annexin V agglutination, DNA fragmentation, cell shrinkage, endoplasmic reticulum dilation, cell fragmentation, and / or formation of membrane vesicles (called apoptotic bodies). The cell is a cellular tumor, for example, a breast, ovary, stomach,
[082] [082] The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal. In the case of cancer, the therapeutically effective amount of the drug can reduce the number of cancer cells; reduce the size of the tumor; inhibit (that is, delay to some extent and, preferably, stop) cancer infiltration into peripheral organs; inhibit (i.e., delay to some extent and, preferably, stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or relieve, to some extent, one or more of the symptoms associated with cancer. To some extent, the drug can inhibit the growth of and / or kill existing cancer cells, and can be cytotoxic or cytostatic. For cancer therapy, effectiveness can, for example, be measured by assessing the time to disease progression (TTP) and / or determining the response rate (RR).
[083] [083] The term "substantial amount" refers to a majority, that is, more than 50% of a population, mixture or sample.
[084] [084] The term "intracellular metabolite" refers to a compound resulting from a metabolite process or reaction within a cell in an antibody-drug conjugate (ADC). The metabolic process or reaction can be an enzymatic process, such as proteolytic cleavage of an ADC peptide ligand. Intracellular metabolites include, but are not limited to, antibodies and free drugs that have undergone intracellular cleavage after entry, diffusion, uptake or transport in a cell.
[085] [085] The terms "intracellularly cleaved" and "intracellular cleavage" refer to a metabolic process or reaction within a cell in an ADC or the like, whereby the covalent bond, for example, the ligand, between the drug and the antibody is broken down, resulting in the free drug, or another metabolite of the conjugate dissociated from the antibody within the cell. The cleaved portions of the ADC are thus intracellular metabolites.
[086] [086] The term "bioavailability" refers to the systemic availability (ie, blood / plasma levels) of a given amount of a drug administered to a patient. Bioavailability is an absolute term that indicates the measurement of both the time (rate) and the total amount (dimension) of the drug reaching the general circulation of an administered dosage form.
[087] [087] The term "cytotoxic activity" refers to an anti-proliferative, cytostatic or cell-killing effect of an ADC or an intracellular metabolite of said ADC. Cytotoxic activity can be expressed as the IC50 value, which is the concentration (molar or mass) per unit volume in which half of the cells survive.
[088] [088] A "disorder" is any condition that may benefit from treatment with a drug or antibody-drug conjugate. This includes chronic or acute disorders or illnesses including those pathological conditions that predispose a mammal to the disorder in question. Non-limiting examples of disorders to be treated here include benign malignant cancers; lymphoid and leukemia malignancies, neuronal, glial, astrocytic, hypothalamic and other glandular, macrophage, epithelial, stromal and blastocele disorders; and inflammatory, angiogenic and immunological disorders.
[089] [089] The terms "cancer" and "cancerous" refer to or describe the condition or physiological disorder in mammals that is typically characterized by unregulated cell growth. A "tumor" comprises one or more cancer cells.
[090] [090] Examples of a "patient" include, but are not limited to, a human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird and chicken. In an exemplary embodiment, the patient is a human being.
[091] [091] The terms "treat" or "treatment," unless otherwise indicated by the context, refer to therapeutic treatment and prophylactic measures to prevent recurrence, where the object is to inhibit or reduce (alleviate) an unwanted physiological disorder or change , such as the development or spread of cancer. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, symptom relief, decrease in the extent of the disease, stabilized state (i.e., not worsening) of disease, delay or decrease in disease progression, improvement or palliation of the disease state, and remission (whether partial or total), if detectable or undetectable.
[092] [092] In the context of cancer, the term "treatment" includes any or all growth inhibition of tumor cells, cancer cells, or a tumor; inhibiting the replication of tumor cells or cancer cells, reducing the total tumor burden or decreasing the number of cancer cells, and improving one or more symptoms associated with the disease.
[093] [093] In the context of an autoimmune disease, the term "treat" includes any or all of the inhibition of cell replication associated with an autoimmune disease state including, but not limited to, cells that produce an autoimmune antibody, reducing the burden of autoimmune antibody and improvement of one or more symptoms of an autoimmune disease.
[094] [094] In the context of an infectious disease, the term "treat" includes any or all: inhibiting the growth, multiplication or replication of the pathogen that causes the infectious disease and ameliorating one or more symptoms of an infectious disease.
[095] [095] The term "package insert" is used to refer to instructions normally included in commercial packaging of therapeutic products, which contain information on the indication (s), use, dosage, administration, contraindications and / or precautions regarding the use of such therapeutic products.
[096] [096] As used here, the terms "cell", "cell line" and "cell culture" are used interchangeably and such designations include descent. The words "transformants" and "transformed cells" include the primary and descending primary cultures and stem cells derived from them with no relation to the number of transfers. It is also understood that all descendants may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant descendants that have the same biological function or activity as researched on the originally transformed cell are included.
[097] [097] Unless otherwise indicated, the term "alkyl" alone or as part of another term refers to a saturated, branched or straight chain hydrocarbon having the indicated number of carbon atoms (for example, "C1-C8" alkyl refers to an alkyl group having 1 to 8 carbon atoms). When the number of carbon atoms is not indicated, the alkyl group has 1 to 8 carbon atoms.
[098] [098] Unless otherwise stated, "alkylene," alone or as part of another term, refers to a cyclic or straight chain or branched hydrocarbon radical, saturated with the defined number of carbon atoms, typically 1-18 carbon atoms , and having two monovalent radical centers derived by removing two hydrogen atoms from the same or two different carbon atoms from a parent alkane. Typical alkylene radicals include, but are not limited to: methylene (-CH2-), 1,2-ethylene -CH2CH2-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (- CH2CH2CH2CH2-), and the like. A "C1-C10" straight chain alkylene is a straight chain saturated hydrocarbon group of the formula - (CH2) 1-10-. Examples of a C1-C10 alkylene include methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, ocytylene, nonylene and decalene.
[099] [099] Unless otherwise specified, the term "heteroalkyl", alone or in combination with another term, means, unless otherwise stated, a stable straight or branched chain hydrocarbon, or combinations thereof, fully saturated or containing from 1 to 3 degrees of unsaturation, which consists of the defined number of carbon atoms, and one to three heteroatoms selected from the group consisting of O, N, Si and S, and in which the sulfur and nitrogen atoms can optionally be oxidized and the nitrogen heteroatom can optionally be quaternized. The heteroatom (s) O, N and S can be placed in any interior position of the heteroalkyl group. The Si heteroatom can be placed anywhere in the heteroalkyl group, including the position where the alkyl group is attached to the rest of the molecule. Up to two heteroatoms can be consecutive.
[0100] [0100] Unless otherwise indicated, the term "heteroalkylene" alone or as part of another substitute means a divalent group derived from heteroalkyl (as discussed above). For heteroalkylene groups, heteroatoms can also occupy one or both chain ends.
[0101] [0101] Unless otherwise indicated, "aryl", alone or a part of another term, means an unsubstituted or substituted monovalent carbocyclic aromatic hydrocarbon radical of 6-20, preferably 6-14, carbon atoms derived by the removal of an atom of hydrogen from a single carbon atom of a relative aromatic ring system. Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, and the like. A substituted aromatic carbocyclic group (for example, an aryl group) can be substituted with one or more, preferably 1 to 5, of the following groups: C1-C8 alkyl, -O- (C1-C8 alkyl), -C (O) R ', -OC (O) R', -C (O) OR ', -C (O) NH2, -C (O) NHR', -C (O) N (R ') 2, -NHC (O ) R ', -S (O) 2R', -S (O) R ', -OH, halogen, -N3, -NH2, -NH (R'), -N (R ') 2 and -CN; wherein each R 'is independently selected from -H, C1-C8 alkyl and unsubstituted aryl. In some embodiments, a substituted carbocyclic aromatic group may further include one or more of: -NHC (= NH) NH2, -NHCONH2, -S (= O) 2R 'and -SR'. "Arylene" is the corresponding divalent portion.
[0102] [0102] "Substituted alkyl" means an alkyl in which one or more hydrogen atoms are each independently substituted with a substitute.
[0103] [0103] Unless otherwise indicated, "aralkyl" alone or part of another term, means an alkyl group, as defined above, replaced with an aryl group, as defined above.
[0104] [0104] Unless otherwise indicated, "C1-C10 heterocyclyl" alone or as part of another term, refers to a monovalent, substituted or unsubstituted monocyclic, bicyclic or tricyclic, aromatic or non-aromatic ring system having from 1 to 10 , preferably 3 to 8, carbon atoms (also referred to as ring members) and one to four heteroatom ring members independently selected from N, O, P or S, and derived by removing a hydrogen atom from an atom of ring of a relative ring system. One or more N, C or S atoms in the heterocyclyl can be oxidized. The ring that includes the heteroatom can be aromatic or non-aromatic. Unless otherwise defined, the heterocyclyl is attached to its group suspended on any heteroatom or carbon atom that results in a stable structure. Representative examples of a C1-C10 heterocyclyl include, but are not limited to, tetrahydrofuranyl, oxetanil, pyranyl, pyrrolidinyl, piperidinyl, piperazinyl, benzofuranyl, benzothiophene, benzothiazolil, indolyl, benzopyrazolyl, pyrrolyl, thiophenyl, thiophenyl, thiophenyl, thiophenyl, thiophenyl, thiol , pyrazolyl, triazolyl, quinolinyl including portions, such as 1,2,3,4-tetrahydro-quinolinyl, pyrimidinyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl, tetrazolyl, epoxide, oxide and BODIPY (substituted or substituted). A C1-C10 heterocyclyl can be substituted with up to seven groups including, but not limited to, C1-C8 alkyl, C1-C8 heteroalkyl, -OR ', aryl, -C (O) R', -OC (O) R ' , -C (O) OR ', -C (O) NH2, -C (O) NHR', -C (O) N (R ') 2, -NHC (O) R', -S (= O) 2R ', -S (O) R', halogen, -N3, -NH2, -NH (R '), -N (R') 2 and -CN; wherein each R 'is independently selected from -H, C1-C8 alkyl, C1-C8 heteroalkyl and aryl. In some embodiments, a substituted heterocyclyl may also include one or more of: -NHC (= NH) NH2, -NHCONH2, -S (= O) 2R 'and -SR'. "Heterocycle" "C1-C10 heterocycle" is the corresponding divalent portion.
[0105] [0105] Unless otherwise indicated, "heteroaralkyl" alone or part of another term, means an alkyl group, as defined above, replaced with an aromatic heterocyclyl group, as defined above. Heteroaralkyl is the corresponding divalent portion.
[0106] [0106] Unless otherwise specified, “C3-C8 carbocyclyl” alone or as part of another term, is a non-aromatic, saturated or unsaturated, substituted or unsubstituted, monovalent 3-, 4-, 5-monocyclic or bicyclic ring -, 6-, 7- or 8- members derived by removing a hydrogen atom from a ring atom from a parent ring system. Representative C3-C8 carbocyclyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl, 1,3-cycloheptadienyl, 1,3,5 -cycloheptatrienyl, cyclooctyl, cyclooctadienyl, bicycles (1.1.1.) pentane, and bicycles (2.2.2.) octane. A C3-C8 carbocyclyl group can be unsubstituted or substituted with up to seven groups including, but not limited to, C1-C8 alkyl, C1-C8 heteroalkyl, -OR ', aryl, -C (O) R', -OC (O ) R ', -C (O) OR', -C (O) NH2, -C (O) NHR ', -C (O) N (R') 2, -NHC (O) R ', -S ( = O) 2R ', -S (= O) R', -OH, -halogen, -N3, -NH2, -NH (R '), -N (R') 2 and -CN; wherein each R 'is independently selected from -H, C1-C8 alkyl, C1-C8 heteroalkyl and aryl. “C3-C8 carbocycle” is the corresponding divalent portion.
[0107] [0107] The term "chiral" refers to molecules that have the non-overlapping property of the corresponding mirror image, although the term "achiral" refers to molecules that are liable to overlap in their corresponding mirror image.
[0108] [0108] The term "stereoisomers" refers to compounds that have identical chemical constitution, but differ with respect to the arrangement of atoms or groups in space.
[0109] [0109] "Diastereomer" refers to a stereoisomer with two or more chirality centers and whose molecules are not mirror images of each other.
[0110] [0110] Stereochemical definitions and conventions used here generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms, McGraw-Hill Book Company, New York (1984); and Eliel and Wilen, Stereochemistry of Organic Compounds, John Wiley & Sons, Inc., New York (1994). Many organic compounds exist in optically active forms, that is, they have the ability to rotate the plane of polarized light by plane. When describing an optically active compound, the prefixes D and L, or R and S, are used to represent the absolute configuration of the molecule on its chiral center (s). The prefixes d and l or (+) and (-) are used to denote the signal of rotation of light polarized by plane by the compound, with (-) or 1 meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical, except that they are mirror images of each other. A specific stereoisomer can also be referred to as an enantiomer, and a mixture of atis isomers is generally called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which can occur where there is no stereoselection or stereospecificity in a chemical process or reaction. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, free from optical activity.
[0111] [0111] An amino acid "derivative" includes an amino acid having substitutions or modifications by covalently bonding a parent amino acid, such as, for example, by alkylation, glycosylation, acetylation, phosphorylation and the like.
[0112] [0112] A "natural amino acid" refers to arginine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, glycine, alanine, histidine, serine, proline, glutamic acid, aspartic acid, threonine, cysteine, methionine, leucine, asparagine, isoleucine and valine, unless otherwise specified by the context.
[0113] [0113] "Protection group" refers to a portion that, when attached to a reaction group in molecule masks, reduces or prevents that reactivity. Examples of protecting groups can be found in T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York, 1999, and Harrison and Harrison et al., Compendium of Synthetic Organic Methods, Vols.
[0114] [0114] Examples of a "hydroxyl protecting group" include, but are not limited to, methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, triisopropyl silyl ether, - butyldimethyl silyl ether, triphenylmethyl silyl ether, acetate ester, substituted esters acetate, pivaloate, benzoate, methanesulfonate and p-toluenesulfonate.
[0115] [0115] "Starting group" refers to a functional group that can be replaced by another functional group. Such starting groups are well known in the art, and examples include, but are not limited to, a halide (e.g., chloride, bromide, iodide), methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl),
[0116] [0116] The phrase "pharmaceutically acceptable salt", as used herein, refers to pharmaceutically acceptable organic or inorganic salts of a compound. The compound typically contains at least one amino group, and therefore acid addition salts can be formed with this amino group. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, malate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and this is pamoate salts -methylene-bis- (2-hydroxy-3-naphthoate)). A pharmaceutically acceptable salt may involve the inclusion of another molecule, such as an acetate ion, a succinate ion or another counterion. The counterion can be any organic or inorganic portion that stabilizes the charge on the parent compound. In addition, a pharmaceutically acceptable salt can have more than one atom charged in its structure. Cases where several charged atoms are part of the pharmaceutically acceptable salt can have multiple counterions. Therefore, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterions.
[0117] [0117] "Pharmaceutically acceptable solvate" or "solvate" refers to an association of one or more solvent molecules and a compound or conjugate of the invention. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.
[0118] [0118] The terms "charge" or "drug charge" or "charge" represent or refer to the average number of charges ("charge" and "charges" are used interchangeably here with "drug" and "drugs") per antibody in a molecule
[0119] [0119] Generally, antibodies do not contain many, if any, free and reactive cysteine groups that can be linked to a drug via a linker. Most of the cysteine thiol residues in the antibodies exist as disulfide binders and must be reduced with a reducing agent, such as dithiothreitol (DTT). The antibody may be subject to denaturation conditions to reveal reactive nucleophilic groups, such as lysine or cysteine. The load (drug / antibody ratio) of an ADC can be controlled in different ways, including: (i) limiting the excess molar drug-binder relative to the antibody, (ii) limiting the temperature or time of the conjugation reaction, and (iii) partial reducing or limiting conditions for modifying cysteine thiol. Where more than one nucleophilic group reacts with a drug-linker, then the resulting product is a mixture of ADC with a distribution of one or more drug portions per antibody. The average number of drugs per antibody can be calculated from the mixture by, for example, double antibody-specific and drug-specific ELISA anti-antibody assay. Individual ADCs can be identified in the mixture by mass spectroscopy, and separated by HPLC, for example, hydrophobic interaction chromatography.
[0120] [0120] Below is a list of abbreviations and definitions that cannot be otherwise defined or described in this application: DMSO (refers to dimethyl sulfoxide), HRMS (refers to high resolution mass spectrometry), DAD (refers to detection of diode matrix), TFA (refers to 2,2,2-trifluoroacetic acid or trifluoroacetic acid), TFF (refers to tangential flow filtration), EtOH (refers to ethanol), MW (refers to molecular weight), HPLC (refers to high performance liquid chromatography), HPLC prep (refers to preparative high performance liquid chromatography), etc. (refers to and so on), trityl (refers to 1,1 ', 1' '- ethane-1,1,1-triyltribenzene), THF (refers to tetrahydrofuran), NHS (refers to 1-hydroxy -2,5-pyrrolidinedione), Cbz (refers to carboxybenzyl), eq. (refers to equivalent), n-BuLi (refers to n-butyllithium), OAc (refers to acetate), MeOH (refers to methanol), i-Pr (refers to isopropyl or propan-2-yl) , NMM (refers to 4-methylmorpholine), and “-“ (in a table, refers to no data currently available).
[0121] [0121] Antibody drug compounds and conjugates thereof
[0122] [0122] One aspect of the invention relates to a compound of formula I: I
[0123] [0123] or a pharmaceutically acceptable salt or solvate thereof, where, independently for each occurrence, 1-2 R3A
[0124] [0124] W is, O, R3A
[0125] [0125] R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl;
[0126] [0126] R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl;
[0127] [0127] R3A and R3B are any of the following:
[0128] [0128] R3A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl; and
[0129] [0129] R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; or
[0130] [0130] R3A and R3B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
[0131] [0131] R4A and R4B are any of the following:
[0132] [0132] R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and
[0133] [0133] R4B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or
[0134] [0134] R4A and R4B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
[0135] [0135] R5 is,,,,,,,,,,,,, C1-C10 heterocyclyl, C3-C8 carbocyclyl and C6-C14 aryl optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from the group consisting of -C1-C8 alkyl, -C1-C8 alkyl-N (R ') 2, -C1-C8 alkyl-C (O) R', -C1-C8 alkyl-C (O) OR '-O- (C1-C8 alkyl), -C (O) R ', -OC (O) R', -C (O) OR ', -C (O) N (R') 2, -NHC (O) R ' , -S (O) 2R ', -S (O) R', -OH, halogen, -N3, -N (R ') 2, -CN, -NHC (= NH) NH2, -NHCONH2, -S ( = O) 2R 'and -SR', where each R 'is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl, or two R' can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; O
[0136] [0136] or R5 is, R13, or
[0137] [0137] R6 is hydrogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -C2-C8 alkynyl or -C1-C8 haloalkyl;
[0138] [0138] R12 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl;
[0139] [0139] R13 is C1-C10 heterocyclyl; and
[0140] [0140] X is O or S;
[0141] [0141] since when R3A is hydrogen X is S.
[0142] [0142] Another aspect of the invention relates to a compound of formula IIa: IIa
[0143] [0143] or a pharmaceutically acceptable salt or solvate thereof, where, independently for each occurrence, 1-2 R3A
[0144] [0144] Y is -C2-C20 alkylene-, -C2-C20 heteroalkylene-; -C3-C8 carbocyclo-, -arylene-, -C3-C8heterocyclo-, -Cl-C10alkylene-arylene-, -arylene-Cl-Cl0Clalkylene-, -Cl- Cl0alkylene- (C3-
[0145] [0145] C8carbocycle) -, - (C3-C8carbocycle) -Cl-C10alkylene-, -Cl-Cl0alkylene- (C3-C8 heterocycle) - or - (C3-C8 heterocycle) -Cl-Cl0alkylene-; H N
[0146] [0146] Z is,,, O O O H N
[0147] [0147] R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl;
[0148] [0148] R3A and R3B are any of the following:
[0149] [0149] R3A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and
[0150] [0150] R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl or halogen; or
[0151] [0151] R3A and R3B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
[0152] [0152] R4A and R4B are any of the following:
[0153] [0153] R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and
[0154] [0154] R4B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or
[0155] [0155] R4A and R4B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
[0156] [0156] R5 is,,,,,,,,,,,,, C1-C10 heterocyclyl, C3-C8 carbocyclyl and C6-C14 aryl optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from the group consisting of -C1-C8 alkyl, -C1-C8 alkyl-N (R ') 2, -C1-C8 alkyl-C (O) R', -C1-C8 alkyl-C (O) OR '-O- (C1-C8 alkyl), -C (O) R ', -OC (O) R', -C (O) OR ', -C (O) N (R') 2, -NHC (O) R ' , -S (O) 2R ', -S (O) R', -OH, halogen, -N3, -N (R ') 2, -CN, -NHC (= NH) NH2, -NHCONH2, -S ( = O) 2R 'and -SR', where each R 'is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl, or two R' can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; O
[0157] [0157] or R5 is, R13, or
[0158] [0158] R6 is hydrogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -C1-C8 haloalkyl;
[0159] [0159] R12 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl;
[0160] [0160] R13 is C1-C10 heterocyclyl; and
[0161] [0161] R7 is independently selected for each occurrence in the group consisting of F, Cl, I, Br, NO2, CN and CF3;
[0162] [0162] R10 is hydrogen, -Cl-Cl0alkyl, -C3-C8carbocyclyl, -aryl, -Cl-C10 heteroalkyl, -C3- C8heterocycle, -Cl-Cl0alkylene-aryl, -arylene-Cl-Cl0Clalkyl, -Cl-Cl0alkylene- ( C3- C8carbocycle), - (C3-C8 carbocycle) -Cl-Cl0alkyl, -Cl-Cl0alkylene- (C3-C8heterocycle), and - (C3-C8 heterocycle) -Cl-Cl0alkyl, where aryl in R10 comprising aryl is optionally replaced by [R7] h;
[0163] [0163] h is 1, 2, 3, 4 or 5; and
[0164] [0164] X is O or S;
[0165] [0165] since when R3A is hydrogen X is S.
[0166] [0166] Another aspect of the invention relates to a compound of formula IIIa: IIIa
[0167] [0167] or a pharmaceutically acceptable salt or solvate thereof, where, independently for each occurrence, 1-2 R3A
[0168] [0168] R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl;
[0169] [0169] R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl;
[0170] [0170] R3A and R3B are any of the following:
[0171] [0171] R3A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and
[0172] [0172] R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl; or
[0173] [0173] R3A and R3B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
[0174] [0174] R4A and R4B are any of the following:
[0175] [0175] R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and
[0176] [0176] R4B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or
[0177] [0177] R4A and R4B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
[0178] [0178] R5 is
[0179] [0179] optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -O- (C1-C8 alkyl), -C (O) R ', -OC (O ) R ', -C (O) OR', -C (O) NH2, -C (O) NHR ', -C (O) N (R') 2, -NHC (O) R ', -S ( O) 2R ', -S (O) R', -OH, halogen, -N3, -NH2, -NH (R '), -N (R') 2, -CN, -NHC (= NH) NH2, -NHCONH2, -S (= O) 2R 'and -SR', where each R 'is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl;
[0180] [0180] R11 is either;
[0181] [0181] Y is -C2-C20 alkylene-, -C2-C20 heteroalkylene-, C3-C8 carbocyclo-, -arylene-, -C3-C8 heterocyclo-, -Cl-C10alkylene-arylene-, -arylene-Cl-Cl0alkylene- , -Cl- Cl0 alkylene- (C3-
[0182] [0182] C8carbocycle) -, - (C3-C8carbocycle) -Cl-C10alkylene-, -Cl-Cl0alkylene- (C3-C8 heterocycle) -, or - (C3-C8 heterocycle) -Cl-Cl0alkylene-; H N
[0183] [0183] R7 is independently selected for each occurrence in the group consisting of F, Cl, I, Br, NO2, CN and CF3;
[0184] [0184] R10 is hydrogen, -Cl-Cl0alkyl, -C3-C8carbocycle, aryl, -Cl-C10heteroalkyl, -C3- C8heterocycle, -Cl-Cl0alkylene-aryl, -arylene-Cl-Cl0Alkyl, -Cl-Cl0alkylene- (C3 - C8carbocycle), - (C3-C8 carbocycle) -Cl-Cl0alkyl, -Cl-Cl0alkylene- (C3-C8heterocycle), and - (C3-C8 heterocycle) -Cl-Cl0alkyl, where aryl in R10 comprising aryl is optionally substituted by [R7] h;
[0185] [0185] h is 1, 2, 3, 4 or 5; and
[0186] [0186] X is O or S.
[0187] [0187] R6 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; and
[0188] [0188] h is 1, 2, 3, 4 or 5.
[0189] [0189] Another aspect of the invention relates to a compound of formula IIb: IIb
[0190] [0190] or a pharmaceutically acceptable salt or solvate thereof, where, independently for each occurrence, 1-2 R3A
[0191] [0191] Y is -C2-C20 alkylene-, -C2-C20 heteroalkylene-, -C3-C8 carbocyclo-, -arylene-, -C3-C8 heterocyclo-, -Cl-C10alkylene-arylene-, -arylene-Cl-Cl0alkylene -, -Cl- Cl0 alkylene- (C3-
[0192] [0192] C8carbocycle) -, - (C3-C8carbocycle) -Cl-C10alkylene-, -Cl-Cl0alkylene- (C3-C8 heterocycle) -, or - (C3-C8 heterocycle) -Cl-Cl0alkylene-; Z is,,, O H L N O H N H
[0193] [0193] L is an antibody;
[0194] [0194] R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl;
[0195] [0195] R3A and R3B are any of the following:
[0196] [0196] R3A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and
[0197] [0197] R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl; or
[0198] [0198] R3A and R3B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
[0199] [0199] R4A and R4B are any of the following:
[0200] [0200] R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and
[0201] [0201] R4B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or
[0202] [0202] R4A and R4B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
[0203] [0203] R5 is,,,,,,,,,,,,, C1-C10 heterocyclyl, C3-C8 carbocyclyl and C6-C14 aryl optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from the group consisting of -C1-C8 alkyl, -C1-C8 alkyl-N (R ') 2, -C1-C8 alkyl-C (O) R', -C1-C8 alkyl-C (O) OR '-O- (C1-C8 alkyl), -C (O) R ', -OC (O) R', -C (O) OR ', -C (O) N (R') 2, -NHC (O) R ' , -S (O) 2R ', -S (O) R', -OH, halogen, -N3, -N (R ') 2, -CN, -NHC (= NH) NH2, -NHCONH2, -S ( = O) 2R 'and -SR', where each R 'is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl, or two R' can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; O
[0204] [0204] or R5 is, R13, or
[0205] [0205] R6 is hydrogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -C1-C8 haloalkyl;
[0206] [0206] R12 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl;
[0207] [0207] R13 is C1-C10 heterocyclyl; and
[0208] [0208] X is O or S;
[0209] [0209] since when R3A is hydrogen X is S.
[0210] [0210] Another aspect of the invention relates to a compound of formula IIIb: IIIb
[0211] [0211] or a pharmaceutically acceptable salt or solvate thereof, where, independently for each occurrence, 1-2 R3A
[0212] [0212] W is, O,
[0213] [0213] R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl;
[0214] [0214] R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl;
[0215] [0215] R3A and R3B are any of the following:
[0216] [0216] R3A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and
[0217] [0217] R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl; or
[0218] [0218] R3A and R3B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
[0219] [0219] R4A and R4B are any of the following:
[0220] [0220] R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and
[0221] [0221] R4B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or
[0222] [0222] R4A and R4B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
[0223] [0223] R5 is
[0224] [0224] optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -O- (C1-C8 alkyl), -C (O) R ', -OC (O ) R ', -C (O) OR', -C (O) NH2, -C (O) NHR ', -C (O) N (R') 2, -NHC (O) R ', -S ( O) 2R ', -S (O) R', -OH, halogen, -N3, -NH2, -NH (R '), -N (R') 2, -CN, -NHC (= NH) NH2, -NHCONH2, -S (= O) 2R 'and -SR', where each R 'is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl; R11 is or;
[0225] [0225] Y is -C2-C20 alkylene-, -C2-C20 heteroalkylene-, -C3-C8 carbocyclo-, -arylene-, -C3-C8 heterocyclo-, -Cl-C10alkylene-arylene-, -arylene-Cl-Cl0alkylene -, -Cl- Cl0 alkylene- (C3-
[0226] [0226] C8carbocycle) -, - (C3-C8carbocycle) -Cl-C10alkylene-, -Cl-Cl0alkylene- (C3-C8 heterocycle) -, or - (C3-C8 heterocycle) -Cl-Cl0alkylene-; Z is,,, O H L N O H N H
[0227] [0227] L is an antibody;
[0228] [0228] X is O or S.
[0229] [0229] Another aspect of the invention relates to a compound of formula IIc: R3B 'R3A' H O H L Z 'R1' N N N N D N R5 R2 'O O X
[0230] [0230] or a pharmaceutically acceptable salt or solvate thereof, where, independently for each occurrence, O O O O H N Y N N H H O O NH
[0231] [0231] Y is -C2-C20 alkylene-, -C2-C20 heteroalkylene-, -C3-C8 carbocyclo-, -arylene-, -C3-C8 heterocyclo-, -Cl-C10alkylene-arylene-, -arylene-Cl-Cl0alkylene -, -Cl- Cl0 alkylene- (C3-
[0232] [0232] C8carbocycle) -, - (C3-C8carbocycle) -Cl-C10alkylene-, -Cl-Cl0alkylene- (C3-C8 heterocycle) -, or - (C3-C8 heterocycle) -Cl-Cl0alkylene-; O H O
[0233] [0233] L is an antibody;
[0234] [0234] D is –C (R4A ') (R4B') - or is absent;
[0235] [0235] R2 'is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, or is absent if present;
[0236] [0236] R3A ’and R3B’ are any of the following:
[0237] [0237] R3A 'is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and
[0238] [0238] R3B 'is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl, or R3B' is C2-C4 alkylene and 5-7 membered ring form as indicated by; or
[0239] [0239] R3A 'and R3B', taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
[0240] [0240] R4A ’and R4B’ are any of the following:
[0241] [0241] R4A 'is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and
[0242] [0242] R4B 'is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or
[0243] [0243] R4A 'and R4B', taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
[0244] [0244] R5 is,,,,,,,,,,,,, C1-C10 heterocyclyl, C3-C8 carbocyclyl and C6-C14 aryl optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from the group consisting of -C1-C8 alkyl, -C1-C8 alkyl-N (R ') 2, -C1-C8 alkyl-C (O) R', -C1-C8 alkyl-C (O) OR '-O- (C1-C8 alkyl), -C (O) R ', -OC (O) R', -C (O) OR ', -C (O) N (R') 2, -NHC (O) R ' , -S (O) 2R ', -S (O) R', -OH, halogen, -N3, -N (R ') 2, -CN, -NHC (= NH) NH2, -NHCONH2, -S ( = O) 2R 'and -SR', where each R 'is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl, or two R' can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl;
[0245] [0245] or R5 is,
[0246] [0246] R6 is hydrogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -C1-C8 haloalkyl;
[0247] [0247] R12 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl;
[0248] [0248] R13 is C1-C10 heterocyclyl; and
[0249] [0249] X is O or S;
[0250] [0250] since when R3A is hydrogen X is S.
[0251] [0251] Another aspect of the invention relates to a compound of formula IIIc: H O H
[0252] [0252] or a pharmaceutically acceptable salt or solvate thereof, where, independently for each occurrence, 1-2 R3A
[0253] [0253] R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl;
[0254] [0254] R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl;
[0255] [0255] R3A and R3B are any of the following:
[0256] [0256] R3A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and
[0257] [0257] R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl; or
[0258] [0258] R3A and R3B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
[0259] [0259] R4A and R4B are any of the following:
[0260] [0260] R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and
[0261] [0261] R4B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or
[0262] [0262] R4A and R4B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
[0263] [0263] R5 is
[0264] [0264] optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -O- (C1-C8 alkyl), -C (O) R ', -OC (O ) R ', -C (O) OR', -C (O) NH2, -C (O) NHR ', -C (O) N (R') 2, -NHC (O) R ', -S ( O) 2R ', -S (O) R', -OH, halogen, -N3, -NH2, -NH (R '), -N (R') 2, -CN, -NHC (= NH) NH2, -NHCONH2, -S (= O) 2R 'and -SR', where each R 'is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl; O O O O H N Y N N H H O O NH
[0265] [0265] Y is -C2-C20 alkylene-, -C2-C20 heteroalkylene-, -C3-C8 carbocyclo-, -arylene-, -C3-C8 heterocyclo-, -Cl-C10alkylene-arylene-, -arylene-Cl-Cl0alkylene -, -Cl- Cl0 alkylene- (C3-
[0266] [0266] C8carbocycle) -, - (C3-C8carbocycle) -Cl-C10alkylene-, -Cl-Cl0alkylene- (C3-C8 heterocycle) -, or - (C3-C8 heterocycle) -Cl-Cl0alkylene-; O H O
[0267] [0267] L is an antibody;
[0268] [0268] X is O or S.
[0269] [0269] Another aspect of the invention relates to a compound of formula IId: R3B 'R3A' H O H L [linker] N N N N D N R5 R2 '
[0270] [0270] or a pharmaceutically acceptable salt or solvate thereof, where, independently for each occurrence,
[0271] [0271] L is an antibody;
[0272] [0272] [linker] is a divalent linker;
[0273] [0273] D is –C (R4A ') (R4B') - or is absent;
[0274] [0274] R2 'is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, or is absent if present;
[0275] [0275] R3A ’and R3B’ are any of the following:
[0276] [0276] R3A 'is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and
[0277] [0277] R3B 'is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl, or R3B' is C2-C4 alkylene and 5-7 membered ring form as indicated by; or
[0278] [0278] R3A 'and R3B', taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
[0279] [0279] R4A ’and R4B’ are any of the following:
[0280] [0280] R4A 'is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and
[0281] [0281] R4B 'is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or
[0282] [0282] R4A 'and R4B', taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
[0283] [0283] R5 is,,,,,,,,,,,,, C1-C10 heterocyclyl, C3-C8 carbocyclyl and C6-C14 aryl optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from the group consisting of -C1-C8 alkyl, -C1-C8 alkyl-N (R ') 2, -C1-C8 alkyl-C (O) R', -C1-C8 alkyl-C (O) OR '-O- (C1-C8 alkyl), -C (O) R ', -OC (O) R', -C (O) OR ',
[0284] [0284] or R5 is, R13, or
[0285] [0285] R6 is hydrogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -C1-C8 haloalkyl;
[0286] [0286] R12 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl;
[0287] [0287] R13 is C1-C10 heterocyclyl; and
[0288] [0288] X is O or S;
[0289] [0289] since when R3A is hydrogen X is S.
[0290] [0290] Another aspect of the invention relates to a compound of formula IIId: H O H
[0291] [0291] or a pharmaceutically acceptable salt or solvate thereof, where, independently for each occurrence, 1-2 R3A
[0292] [0292] R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl;
[0293] [0293] R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl;
[0294] [0294] R3A and R3B are any of the following:
[0295] [0295] R3A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and
[0296] [0296] R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl; or
[0297] [0297] R3A and R3B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
[0298] [0298] R4A and R4B are any of the following:
[0299] [0299] R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and
[0300] [0300] R4B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or
[0301] [0301] R4A and R4B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
[0302] [0302] R5 is
[0303] [0303] optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -O- (C1-C8 alkyl), -C (O) R ', -OC (O ) R ', -C (O) OR', -C (O) NH2, -C (O) NHR ', -C (O) N (R') 2, -NHC (O) R ', -S ( O) 2R ', -S (O) R', -OH, halogen, -N3, -NH2, -NH (R '), -N (R') 2, -CN, -NHC (= NH) NH2, -NHCONH2, -S (= O) 2R 'and -SR', where each R 'is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl;
[0304] [0304] [linker] is a divalent linker;
[0305] [0305] L is an antibody;
[0306] [0306] X is O or S.
[0307] [0307] In certain embodiments, the present invention relates to any of the above-mentioned compounds and their definitions, in which the compound is represented by.
[0308] [0308] In certain embodiments, the present invention relates to any of the above-mentioned compounds and their definitions, where W is.
[0309] [0309] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where W is 1-2 R3A
[0310] [0310] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where W is.
[0311] [0311] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, in which R3A
[0312] [0312] W is either O.
[0313] [0313] In certain embodiments of the invention, W is:
[0314] [0314] In certain embodiments, the present invention relates to any of the above-mentioned compounds and their definitions, wherein R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl.
[0315] [0315] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R1 is hydrogen.
[0316] [0316] In certain embodiments, the present invention relates to any of the aforementioned compounds and their definitions, wherein R1 is C1-C8 alkyl.
[0317] [0317] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R1 is methyl.
[0318] [0318] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, wherein R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl.
[0319] [0319] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R2 is hydrogen.
[0320] [0320] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, wherein R2 is C1-C8 alkyl.
[0321] [0321] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R2 is methyl.
[0322] [0322] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R1 is hydrogen; and R2 is methyl.
[0323] [0323] In certain embodiments, the present invention relates to any of the aforementioned compounds and their definitions, wherein R1 is methyl; and R2 is methyl.
[0324] [0324] In certain embodiments, the present invention relates to any of the aforementioned compounds and their definitions, wherein R3A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl.
[0325] [0325] In certain embodiments, the present invention relates to any of the aforementioned compounds and their definitions, wherein R3A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen.
[0326] [0326] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R3A is halogen.
[0327] [0327] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R3A is hydrogen.
[0328] [0328] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, wherein R3A is C1-C8 alkyl.
[0329] [0329] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R3A is methyl.
[0330] [0330] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, wherein R3B is C1-C8 alkyl.
[0331] [0331] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R3B is methyl.
[0332] [0332] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, wherein R3B is isopropyl.
[0333] [0333] In certain embodiments, the present invention relates to any of the above-mentioned compounds and their definitions, wherein R3B is C3-C8 carbocyclyl.
[0334] [0334] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, wherein R3B is cyclohexyl.
[0335] [0335] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, wherein R3A is C1-C8 alkyl; and R3B is C1-C8 alkyl.
[0336] [0336] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, wherein R3A is methyl; and R3B is methyl.
[0337] [0337] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R3A is hydrogen; and R3B is C1-C8 alkyl.
[0338] [0338] In certain embodiments, the present invention relates to any of the aforementioned compounds and their definitions, wherein R3A is hydrogen; and R3B is isopropyl.
[0339] [0339] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, wherein R3A and R3B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene.
[0340] [0340] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, wherein R3A and R3B, taken together, are C2-C8 alkylene.
[0341] [0341] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, in which R3A and R3B, taken together, are –CH2CH2–.
[0342] [0342] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, in which R3A and R3B, taken together, are –CH2CH2CH2–.
[0343] [0343] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, in which R3A and R3B, taken together, are –CH2CH2CH2CH2–.
[0344] [0344] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, wherein R3A and R3B, taken together, are C1-C8 heteroalkylene.
[0345] [0345] In certain embodiments, the present invention relates to any of the aforementioned compounds and their definitions, in which R3A and R3B, taken together, are –CH2OCH2–.
[0346] [0346] In certain embodiments, the present invention relates to any of the aforementioned compounds and their definitions, wherein R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R4B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl.
[0347] [0347] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R4A is hydrogen.
[0348] [0348] In certain embodiments, the present invention relates to any of the aforementioned compounds and their definitions, wherein R4A is C1-C8 alkyl.
[0349] [0349] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R4A is methyl.
[0350] [0350] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R4B is hydrogen.
[0351] [0351] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, wherein R4B is C1-C8 alkyl.
[0352] [0352] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R4B is methyl.
[0353] [0353] In certain embodiments, the present invention relates to any of the above-mentioned compounds and their definitions, wherein R4A is C1-C8 alkyl; and R4B is C1-C8 alkyl.
[0354] [0354] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, wherein R4A is methyl; and R4B is methyl.
[0355] [0355] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R4A is hydrogen; and R4B is hydrogen.
[0356] [0356] In certain embodiments, the present invention relates to any of the aforementioned compounds and their definitions, wherein R4A is hydrogen; and R4B is C1-C8 alkyl.
[0357] [0357] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, wherein R4A and R4B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene.
[0358] [0358] In certain embodiments, the present invention relates to any of the aforementioned compounds and their definitions, wherein R4A and R4B, taken together, are C2-C8 alkylene.
[0359] [0359] In certain embodiments, the present invention relates to any of the aforementioned compounds and their definitions, in which R4A and R4B, taken together, are –CH2CH2–.
[0360] [0360] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, in which R4A and R4B, taken together, are –CH2CH2CH2–.
[0361] [0361] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, in which R4A and R4B, taken together, are –CH2CH2CH2CH2–.
[0362] [0362] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, wherein R4A and R4B, taken together, are C1-C8 heteroalkylene.
[0363] [0363] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, in which R4A and R4B, taken together, are -CH2OCH2–.
[0364] [0364] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R5 is.
[0365] [0365] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R5 is.
[0366] [0366] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R5 is.
[0367] [0367] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R5 is.
[0368] [0368] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R5 is.
[0369] [0369] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R5 is.
[0370] [0370] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R5 is.
[0371] [0371] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R5 is.
[0372] [0372] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R5 is.
[0373] [0373] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R5 is.
[0374] [0374] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R5 is.
[0375] [0375] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R5 is.
[0376] [0376] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R5 is.
[0377] [0377] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R5 is.
[0378] [0378] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R5 is
[0379] [0379] In certain embodiments, the present invention relates to any of the above-mentioned compounds and their definitions, where R5 is.
[0380] [0380] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R5 is.
[0381] [0381] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R6 is hydrogen.
[0382] [0382] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, wherein R6 is C1-C8 alkyl.
[0383] [0383] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R6 is methyl.
[0384] [0384] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where X is O.
[0385] [0385] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where X is S.
[0386] [0386] In certain embodiments, the present invention relates to any of the aforementioned compounds, or a pharmaceutically acceptable salt or solvate thereof, and definitions thereof, wherein the compound is selected from the group consisting of:
[0387] [0387] N-Methyl-L-valyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-
[0388] [0388] N2 - [(1-Aminocyclopentyl) carbonyl] -N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) - 1-methoxy-2 -methyl-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} -3-thioxopropyl] pyrrolidin-1-yl} -5-methyl-1- oxoheptan-4-yl] -N-methyl-L-valinamide;
[0389] [0389] 2-Methylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3- {[ (1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} -3-thioxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] - N-methyl-L-valinamide;
[0390] [0390] N-Methyl-L-valyl-N - {(3R, 4S, 5S) -3-methoxy-1 - [(2S) -2 - {(1R, 2R) -1-methoxy-2-methyl- 3 - [(2-phenylethyl) amino] -3-thioxopropyl} pyrrolidin-1-yl] -5-methyl-1-oxoheptan-4-yl} -N-methyl-L-valinamide;
[0391] [0391] N-Methyl-L-valyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-carboxy- 2-phenylethyl] amino} -1-methoxy-2-methyl-3-thioxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide ;
[0392] [0392] N-Methyl-L-valyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-3 - {[ (2S) - 1-methoxy-1-oxo-3-phenylpropan-2-yl] amino} -2-methyl-3-thioxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide;
[0393] [0393] 2-Methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-carboxy-2-phenylethyl] amino} -1-methoxy-2-methyl-3-thioxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide;
[0394] [0394] 2-Methylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-3 - {[(2S) - 1- methoxy-1-oxo-3-phenylpropan-2-yl] amino} -2-methyl-3-thioxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl -L-valinamide;
[0395] [0395] N2 - [(1-Aminocyclopentyl) carbonyl] -N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) - 1-methoxy-2 -methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} -5-methyl-1 -oxoheptan-4-yl] -N-methyl-L-valinamide;
[0396] [0396] N2 - [(1-Aminocyclopropyl) carbonyl] -N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -
[0397] [0397] 1-Amino-N - [(2S) -1 - {[(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy- 2- methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} -5-methyl- 1-oxoheptan-4-yl] (methyl) amino} -3-methyl-1-oxobutan-2-yl] cyclohexanecarboxamide;
[0398] [0398] 2-Methylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3-oxo- 3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide;
[0399] [0399] 2-Methylalanyl-N - {(3R, 4S, 5S) -3-methoxy-1 - [(2S) -2 - {(1R, 2R) -1-methoxy-2-methyl-3-oxo- 3 - [(2-phenylethyl) amino] propyl} pyrrolidin-1-yl] -5-methyl-1-oxoheptan-4-yl} -N-methyl-L-valinamide;
[0400] [0400] 2-Methylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3-oxo- 3 - {[(1-phenylcyclopropyl) methyl] amino} propyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide;
[0401] [0401] 2-Methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[2- (cyclohepta-2,4,6-trien -1- yl) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl- L-valinamide;
[0402] [0402] 2-Methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-carboxy-2-phenylethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide;
[0403] [0403] 2-Methylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-3 - {[(2S) - 1- methoxy-1-oxo-3-phenylpropan-2-yl] amino} -2-methyl-3-oxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl -L-valinamide;
[0404] [0404] N2 - [(3-Aminooxetan-3-yl) carbonyl] -N - {(3R, 4S, 5S) -3-methoxy-1 - [(2S) -2- {(1R, 2R) -1 -methoxy-2-methyl-3-oxo-3 - [(2-phenylethyl) amino] propyl} pyrrolidin-1-yl] -5-methyl-1-oxoheptan-4-yl} -N-methyl-L-valinamide ;
[0405] [0405] N, 2-Dimethylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-3-
[0406] [0406] N, 2-dimethylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-carboxy-2- phenylethyl] amino} -1-methoxy-2-methyl-3-thioxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide;
[0407] [0407] N, 2-Dimethylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3- {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} -3-thioxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl ] -N-methyl-L-valinamide;
[0408] [0408] N, 2-Dimethylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3- oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4- yl] -N-methyl-L-valinamide;
[0409] [0409] N2- (3-Amino-2,2-dimethylpropanoyl) -N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2- [(1R, 2R) -1- methoxy-2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} -5- methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide;
[0410] [0410] N2- (3-Amino-2,2-dimethylpropanoyl) -N - {(3R, 4S, 5S) -3-methoxy-1 - [(2S) -2- {(1R, 2R) -1- methoxy-2-methyl-3-oxo-3 - [(2-phenylethyl) amino] propyl} pyrrolidin-1-yl] -5-methyl-1-oxoheptan-4-yl} -N-methyl-L-valinamide;
[0411] [0411] 2-Methyl-L-prolyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl- 3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan- 4-yl] -N-methyl-L-vaLinamide;
[0412] [0412] 2-Methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[1- (bicycle [4.2.0] octa-1 , 3,5-trien-7-yl) -2-methoxy-2-oxoethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3- methoxy-5-methyl- 1-oxoheptan-4-yl] -N-methyl-L-valinamide;
[0413] [0413] 2-Methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[bicycle [4.2.0] octa-1,3, 5- trien-7-yl (carboxy) methyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide;
[0414] [0414] 2-Methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(1S, 2R) -1-hydroxy-1-
[0415] [0415] N, 2-dimethylalanyl-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-carboxy-2-phenylethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy-1 - [(1S) -1- methylpropyl] -4-oxobutyl} -N-methyl-L-valinamide ;
[0416] [0416] 2-methyl-L-prolyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-3 - {[ (2S) - 1-methoxy-1-oxo-3-phenylpropan-2-yl] amino} -2-methyl-3 oxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] - N-methyl-L-valinamide;
[0417] [0417] 2-methyl-L-prolyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-carboxy- 2-phenylethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide ;
[0418] [0418] 2-methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(2S) -1-tert-butoxy-1- oxo- 3-phenylpropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-valinamide;
[0419] [0419] Methyl N - [(2R, 3R) -3 - {(2S) -1 - [(3R, 4S, 5S) -4 - {[N- (3-amino-2,2-dimethylpropanoyl) - L -valyl] (methyl) amino} -3-methoxy-5-methylheptanoyl] pyrrolidin-2-yl} -3-methoxy-2-methylpropanoyl] -L-phenylalaninate;
[0420] [0420] N, 2-dimethylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(2S) -1-tert-butoxy- 1- oxo-3-phenylpropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide;
[0421] [0421] 2-methyl-D-prolyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-3 - {[ (2S) - 1-methoxy-1-oxo-3-phenylpropan-2-yl] amino} -2-methyl-3-oxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide;
[0422] [0422] 2-methyl-L-prolyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(1S, 2R) -1- hydroxy-1-phenylpropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-valinamide;
[0423] [0423] N, 2-dimethylalanyl-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-benzyl-2- (methylamino ) -2-oxoethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy-1- [(1S) -1-methylpropyl] -4-oxobutyl} -N -methyl-L-valinamide;
[0424] [0424] N, 2-dimethylalanyl-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -2-amino-1-benzyl- 2- oxoethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy-1 - [(1S) -1- methylpropyl] -4-oxobutyl} -N-methyl -L-valinamide;
[0425] [0425] N, 2-dimethylalanyl-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-benzyl-2-oxo- 2- (propylamino) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy-1 - [(1S) - 1-methylpropyl] -4-oxobutyl} - N-methyl-L-valinamide;
[0426] [0426] N, 2-dimethylalanyl-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-benzyl-2- (diethylamino ) -2-oxoethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy-1- [(1S) -1-methylpropyl] -4-oxobutyl} -N -methyl-L-valinamide;
[0427] [0427] N, 2-dimethylalanyl-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-benzyl-2- (tert - butylamino) -2-oxoethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy-1- [(1S) -1-methylpropyl] -4-oxobutyl} -N-methyl-L-valinamide;
[0428] [0428] N, 2-dimethylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(1S, 2R) -1-hydroxy- 1- phenylpropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl -L-valinamide;
[0429] [0429] 3-methyl-D-isovalyl-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-benzyl-2- methoxy-2-oxoethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy-1 - [(1S) -1- methylpropyl] -4-oxobutyl} -N -methyl-L-valinamide;
[0430] [0430] 3-methyl-L-isovalyl-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-benzyl-2- methoxy-2-oxoethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy-1 - [(1S) -1- methylpropyl] -4-oxobutyl} -N -methyl-L-valinamide;
[0431] [0431] L-isovalyl-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-benzyl-2-methoxy-2- oxoethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy-1 - [(1S) -1- methylpropyl] -4-oxobutyl} -N-methyl-L -valinamide;
[0432] [0432] D-isovalyl-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-benzyl-2-methoxy-2- oxoethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy-1 - [(1S) -1- methylpropyl] -4-oxobutyl} -N-methyl-L -valinamide;
[0433] [0433] 1,2-dimethyl-L-prolyl-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-carboxy 2- phenylethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy-1 - [(1S) -1- methylpropyl] -4-oxobutyl} -N-methyl -L-valinamide;
[0434] [0434] 1,2-dimethyl-D-prolyl-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-carboxy- 2- phenylethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy-1 - [(1S) -1- methylpropyl] -4-oxobutyl} -N-methyl -L-valinamide;
[0435] [0435] N ~ 2 ~ - [2,2-dimethyl-3- (methylamino) propanoyl] -N - {(1S, 2R) -2-methoxy-4 - {(2S) -2- [(1R, 2R ) -1-methoxy-2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl } -1 - [(1S) -1-methylpropyl] -4-oxobutyl} -N-methyl-L-valinamide;
[0436] [0436] Methyl N - {(2R, 3R) -3 - [(2S) -1 - {(3R, 4S, 5S) -4 - [{N- [2,2-dimethyl-3- (methylamino) propanoyl ] -L-valyl} (methyl) amino] -3-methoxy-5-methylheptanoyl} pyrrolidin-2-yl] -3-methoxy-2-methylpropanoyl} -L-phenylalaninate;
[0437] [0437] Methyl N - {(2R, 3R) -3-methoxy-3 - [(2S) -1 - {(3R, 4S, 5S) -3-methoxy-5-methyl-4- [methyl (N- {[(2S) -2-methylpiperidin-2-yl] carbonyl} -L-valyl) amino] heptanoyl} pyrrolidin-2-yl] -2-methylpropanoyl} -L-phenylalaninate;
[0438] [0438] Methyl N - {(2R, 3R) -3-methoxy-3 - [(2S) -1 - {(3R, 4S, 5S) -3-methoxy-5-methyl-4- [methyl (N- {[(2R) -2-methylpiperidin-2-yl] carbonyl} -L-valyl) amino] heptanoyl} pyrrolidin-2-yl] -2-methylpropanoyl} -L-phenylalaninate;
[0439] [0439] N - {(2R, 3R) -3-methoxy-3 - [(2S) -1 - {(3R, 4S, 5S) -3-methoxy-5-methyl-4- [methyl (N- { [(2S) -2-methylpiperidin-2-yl] carbonyl} -L-valyl) amino] heptanoyl} pyrrolidin-2-yl] -2-methylpropanoyl} -L-phenylalanine;
[0440] [0440] N - {(2R, 3R) -3-methoxy-3 - [(2S) -1 - {(3R, 4S, 5S) -3-methoxy-5-methyl-4- [methyl (N- { [(2R) -2-methylpiperidin-2-yl] carbonyl} -L-valyl) amino] heptanoyl} pyrrolidin-2-yl] -2-methylpropanoyl} -L-phenylalanine;
[0441] [0441] Methyl N - {(2R, 3R) -3 - [(2S) -1 - {(3R, 4S, 5S) -4 - [(N - {[(3R) -3-fluoropyrrolidin-3-yl ] carbonyl} -L-valyl) (methyl) amino] -3-methoxy-5-methylheptanoyl} pyrrolidin-2-yl] -3-methoxy-2-methylpropanoyl} -L-phenylalaninate;
[0442] [0442] Methyl N - {(2R, 3R) -3 - [(2S) -1 - {(3R, 4S, 5S) -4 - [(N - {[(3R) -3-fluoropyrrolidin-3-yl ] carbonyl} -L-valyl) (methyl) amino] -3-methoxy-5-methylheptanoyl} pyrrolidin-2-yl] -3-methoxy-2-methylpropanoyl} -L-phenylalaninate;
[0443] [0443] (2S) -N - [(2S) -1 - {[(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[2- (cyclohepta -2,4,6-trien- 1-yl) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4 -yl] (methyl) amino} -3-methyl-1-oxobutan-2-yl] -2-methylpiperidine-2-carboxamide;
[0444] [0444] (2R) -N - [(2S) -1 - {[(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[2- (cyclohepta -2,4,6-trien- 1-yl) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4 -yl] (methyl) amino} -3-methyl-1-oxobutan-2-yl] -2-methylpiperidine-2-carboxamide; i. 2-methyl-L-prolyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[2- (cyclohepta-2,4,6- trien-1-yl) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl -L-valinamide;
[0445] [0445] N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[2- (cyclohepta-2,4,6-trien-1-yl ) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N ~ 2 ~ - {[( 3R) -3-fluoropyrrolidin-3-yl] carbonyl} -N-methyl-L-valinamide;
[0446] [0446] N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[2- (cyclohepta-2,4,6-trien-1-yl ) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N ~ 2 ~ - {[( 3S) -3-fluoropyrrolidin-3-yl] carbonyl} -N-methyl-L-valinamide;
[0447] [0447] (2S) -N - [(2S) -1 - {[(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy- 2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} -5-methyl- 1-oxoheptan-4-yl] (methyl) amino} -3-methyl-1-oxobutan-2-yl] -2-methylpiperidine-2-carboxamide;
[0448] [0448] (2R) -N - [(2S) -1 - {[(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy- 2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} -5-methyl- 1-oxoheptan-4-yl] (methyl) amino} -3-methyl-1-oxobutan-2-yl] -2-methylpiperidine-2-carboxamide;
[0449] [0449] N-2 - {[(3R) -3-fluoropyrrolidin-3-yl] carbonyl} -N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2- [( 1R, 2R) -1-methoxy-2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin- 1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide;
[0450] [0450] N-2 - {[(3S) -3-fluoropyrrolidin-3-yl] carbonyl} -N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2- [( 1R, 2R) -1-methoxy-2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin- 1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide;
[0451] [0451] 1,2-dimethyl-D-prolyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(2S) -3- (4-aminophenyl) -1-methoxy-1-oxopropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1- oxoheptan-4-yl] -N-methyl-L-valinamide;
[0452] [0452] 1,2-dimethyl-D-prolyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(2S) -3- (4-aminophenyl) -1-methoxy-1-oxopropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1- oxoheptan-4-yl] -N-methyl-L-valinamide;
[0453] [0453] 1,2-dimethyl-L-prolyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-3- {[(2S) -1-methoxy-1-oxo-3- (1,2,3,4-tetrahydroquinolin-6-yl) propan-2-yl] amino} -2-methyl-3-oxopropyl] pyrrolidin- 1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide;
[0454] [0454] 1,2-dimethyl-L-prolyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(2S) -3- (4-aminophenyl) -1-methoxy-1-oxopropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1- oxoheptan-4-yl] -N-methyl-L-valinamide;
[0455] [0455] 1,2-dimethyl-L-prolyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-3- {[(2S) -1-methoxy-1-oxo-3-phenylpropan-2-yl] amino} -2-methyl-3-oxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4- yl] -N-methyl-L-valinamide;
[0456] [0456] N - {(2R, 3R) -3 - [(2S) -1 - {(3R, 4S, 5S) -4 - [(N - {[(3R) -3-fluoropyrrolidin-3-yl] carbonyl} - L-valyl) (methyl) amino] -3-methoxy-5-methylheptanoyl} pyrrolidin-2-yl] -3-methoxy-2-methylpropanoyl} -L-phenylalanine;
[0457] [0457] N - {(2R, 3R) -3 - [(2S) -1 - {(3R, 4S, 5S) -4 - [(N - {[(3S) -3-fluoropyrrolidin-3-yl] carbonyl} - L-valyl) (methyl) amino] -3-methoxy-5-methylheptanoyl} pyrrolidin-2-yl] -3-methoxy-2-methylpropanoyl} -L-phenylalanine;
[0458] [0458] 2-methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(2R, 4S) -4-carboxy-1- phenylpentan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L -valinamide;
[0459] [0459] 2-methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3- (bicycle [1.1.1] pent-1-ylamino) - 1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide;
[0460] [0460] 2-methylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-3 - {[(1R) - 2- methoxy-2-oxo-1- (1-phenylcyclopropyl) ethyl] amino} -2-methyl-3-oxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N- methyl-L-valinamide;
[0461] [0461] 2-methylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-3 - {[(1S) - 2- methoxy-2-oxo-1- (1-phenylcyclopropyl) ethyl] amino} -2-methyl-3-oxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N- methyl-L-valinamide;
[0462] [0462] 2-methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - ({(1R) -1 - [(7R) - bicycle [4.2.0] octa-1,3,5-trien-7-yl] -2-methoxy-2-oxoethyl} amino) -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} - 3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide;
[0463] [0463] 2-methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - ({(1S) -1 - [(7S) - bicycle [4.2.0] octa-1,3,5-trien-7-yl] -2-methoxy-2-oxoethyl} amino) -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} - 3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide;
[0464] [0464] N, 2-dimethylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-3- {[(2S ) -1-methoxy-1-oxo-3-phenylpropan-2-yl] amino} -2-methyl-3-oxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N -methyl-L-valinamide;
[0465] [0465] 2-methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - ({(1S) -1 - [(7R) - bicycle [4.2.0] octa-1,3,5-trien-7-yl] -2-methoxy-2-oxoethyl} amino) -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} - 3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide;
[0466] [0466] N, N, 2-trimethylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-3- {[ (2S) -1-methoxy-1-oxo-3-phenylpropan-2-yl] amino} -2-methyl-3-oxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide;
[0467] [0467] N, N, 2-trimethylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-carboxy- 2-phenylethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide ;
[0468] [0468] 2-methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(R) -carboxy (1-phenylcyclopropyl) methyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide;
[0469] [0469] difluoro {2-methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(3R, 4R, 7S) -7-benzyl-15- {2- [(3 , 5-dimethyl-1H-pyrrol-2-yl-kappaN) methylidene] -2H-pyrrol-5-yl-kappaN} -4-methyl-5,8,13-trioxo-2-oxa-6,9,12 -triazapentadecan-3-yl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamidate} boron;
[0470] [0470] 2-methyl-D-prolyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl- 3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan- 4-yl] -N-methyl-L-valinamide;
[0471] [0471] Methyl N - {(2R, 3R) -3 - [(2S) -1 - {(3R, 4S, 5S) -4 - [{N - [(3-aminooxetan-3-yl) carbonyl] - L-valyl} (methyl) amino] -3-methoxy-5-methylheptanoyl} pyrrolidin-2-yl] -3-methoxy-2-methylpropanoyl} -L-phenylalaninate;
[0472] [0472] 2-methylalanyl-N - {(3R, 4S, 5S) -1 - [(2S) -2 - {(3R, 4R, 7S, 12S) -7-benzyl-14- [3-chloro-4 - (propan-2-yloxy) phenyl] -4-methyl-12- [4- (8-methylimidazo [1,2-a] pyridin-2-yl) benzyl] -5,8,14-
[0473] [0473] 2-methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(2S) -1 - {[4- (5 -fluoro-1,3-benzothiazol-2-yl) -2-methylphenyl] amino} -1-oxo-3-phenylpropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin- 1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide;
[0474] [0474] 1,2-dimethyl-D-prolyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-3- {[(2S) -1-methoxy-1-oxo-3-phenylpropan-2-yl] amino} -2-methyl-3-oxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4- yl] -N-methyl-L-valinamide;
[0475] [0475] N, 2-dimethylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(2S) -3- (1H-indole -3-yl) - 1-methoxy-1-oxopropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3- methoxy-5-methyl-1- oxoheptan-4-yl] -N-methyl-L-valinamide;
[0476] [0476] N, 2-dimethylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3- oxo-3 - {[(2S) -1-oxo-3-phenyl-1- (prop-2-en-1-yloxy) propan-2-yl] amino} propyl] pyrrolidin-1-yl} -5- methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide;
[0477] [0477] 2-methyl-L-prolyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(2S) -1-tert- butoxy-1-oxo-3-phenylpropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4- yl] -N-methyl-L-valinamide;
[0478] [0478] N, 2-dimethylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3- oxo-3 - ({(2S) -1-oxo-3-phenyl-1 - [(1H-1,2,3-triazol-4-ylmethyl) amino] propan-2-yl} amino) propyl] pyrrolidin- 1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide;
[0479] [0479] N, 2-dimethylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3- oxo-3 - {[(2S) -1-oxo-3-phenyl-1- (prop-2-yn-1-ylamino) propan-2-yl] amino} propyl] pyrrolidin-1-yl} -5- methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide;
[0480] [0480] N, 2-dimethylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(2S) -3- (1H-imidazole -4- yl) -1-methoxy-1-oxopropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1- oxoheptan-4-yl] -N-methyl-L-valinamide;
[0481] [0481] N, 2-dimethylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(2S) -3- (4-hydroxyphenyl ) - 1-methoxy-1-oxopropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4- yl] -N-methyl-L-valinamide;
[0482] [0482] N, 2-dimethylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(1R) -1-carboxy-2- phenylethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide;
[0483] [0483] 1,2-dimethyl-L-prolyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2- methyl-3-oxo-3 - {[(2S) -1-oxo-3-phenyl-1- (piperazin-1-yl) propan-2-yl] amino} propyl] pyrrolidin-1-yl} -5- methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide;
[0484] [0484] 1,2-dimethyl-L-prolyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(2S) -1- amino-3-phenylpropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-valinamide; and
[0485] [0485] 2-methyl-D-prolyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[2- (cyclohepta-2,4 , 6- trien-1-yl) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] - N-methyl-L-valinamide.
[0486] [0486] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R1 is Y Z.
[0487] [0487] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R1 is.
[0488] [0488] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R1 is
[0489] [0489] In certain embodiments, the present invention relates to any of the aforementioned compounds and their definitions, where Y is C2-C20 alkylene.
[0490] [0490] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where Y is - (CH2) p–; and p is 1-10.
[0491] [0491] In certain embodiments, the present invention relates to any of the above-mentioned compounds and their definitions, where p is 1. In certain embodiments, the present invention relates to any of the above-mentioned compounds and their definitions. , where p is 2. In certain embodiments, the present invention relates to any of the aforementioned compounds and their definitions, where p is 3. In certain embodiments, the present invention relates to any of the above compounds mentioned and their definitions, where p is 4. In certain embodiments, the present invention refers to any of the above mentioned compounds and their definitions, where p is 5. In certain embodiments, the present invention relates to any one of the above-mentioned compounds and their definitions, where p is 6. In certain embodiments, the present invention relates to any of the above-mentioned compounds and their definitions, where p is 7. In certain embodiments , the present invention relates to any of the above-mentioned compounds and their definitions, where p is 8. In certain embodiments, the present invention relates to any of the above-mentioned compounds and their definitions, where p is 9 In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where p is 10.
[0492] [0492] In certain embodiments, the present invention relates to any of the aforementioned compounds and their definitions, where Y is C2-C20 heteroalkylene.
[0493] [0493] In certain embodiments, the present invention relates to any of the aforementioned compounds and their definitions, where Y is - (CH- 2CH2O) qCH2CH2–; and q is 1-10.
[0494] [0494] In certain embodiments, the present invention relates to any of the above-mentioned compounds and their definitions, where q is 1. In certain embodiments, the present invention relates to any of the above-mentioned compounds and their definitions. , where q is 2. In certain embodiments, the present invention relates to any of the aforementioned compounds and their definitions, where q is 3. In certain embodiments, the present invention relates to any of the above compounds mentioned and their definitions, where q is 4. In certain embodiments, the present invention relates to any of the aforementioned compounds and their definitions, where q is 5. In certain embodiments, the present invention relates to any one of the above-mentioned compounds and their definitions, where q is 6. In certain embodiments, the present invention relates to any of the above-mentioned compounds and their definitions, where q is 7. In certain embodiments , the present invention relates to any of the above mentioned compounds and their definitions, where q is 8. In certain embodiments, the present invention relates to any of the above mentioned compounds and their definitions, where q is 9 In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where q is 10.
[0495] [0495] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where Z is.
[0496] [0496] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where Z is.
[0497] [0497] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where Z is.
[0498] [0498] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where Z is
[0499] [0499] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where Z is H N AT THE O O .
[0500] [0500] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where Z is O H N
[0501] [0501] In certain embodiments, the present invention relates to any of the aforementioned compounds and their definitions, wherein R7 is F or Cl; and h is 4 or 5.
[0502] [0502] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where R7 is F; and h is 3, 4 or 5.
[0503] [0503] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, wherein R7 is F; and h is 5.
[0504] [0504] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where Z is -NH2.
[0505] [0505] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where G is Cl. In certain embodiments, the present invention relates to any of the above-mentioned compounds and their definitions, where G is Br. In certain embodiments, the present invention relates to any of the above-mentioned compounds and their definitions, where G is I.
[0506] [0506] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, wherein the compound is selected from the group consisting of the compounds of Table 18B.
[0507] [0507] In certain embodiments, the present invention relates to any of the aforementioned compounds and their definitions, wherein the compound is selected from the group consisting of: O O H
[0508] [0508], O O H
[0509] [0509], O O H
[0510] [0510] O O O O O, O O H
[0511] [0511] O O O O O, O O O
[0512] [0512] and O O O
[0513] [0513].
[0514] [0514] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where Z is.
[0515] [0515] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where Z is.
[0516] [0516] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where Z is.
[0517] [0517] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where Z is -NHL.
[0518] [0518] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where Z H N H
[0519] [0519] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where L is H (C) -.
[0520] [0520] In certain embodiments, the present invention relates to any of the compounds mentioned above and their definitions, where L is an antibody selected from a murine antibody for the treatment of ovarian cancer, such as oregovomab (OVAREX® ); a murine IgG2a antibody for the treatment of colorectal cancer, such as edrecolomab (PANOREX®); a chimeric anti-EGFR IgG antibody for the treatment of positive cancers for epidermal growth factor, such as head and neck cancer, for example cetuximab (ERBITUX®); a humanized antibody for the treatment of sarcoma, such as a Humanized Monoclonal Antibody to the Vitronectin Receptor (v 3) such as Vitaxin®; a humanized IgG1 antibody for the treatment of chronic lymphocytic leukemia (CLL), such as alemtuzumab (CAMPATH I / H®); SMART ID10 which is humanized anti-HLA-DR antibody for the treatment of non-Hodgkin's lymphoma; 131I Lym-1 (ONCOLYM®) which is a radio-labeled murine anti-HLA-Dr10 antibody for the treatment of non-lymphoma -Hodgkin; a humanized anti-CD2 mAb for the treatment of Hodgkin's disease or non-Hodgkin's lymphoma, such as ALLOMUNE®; labetuzumab (CEACIDE®) which is a humanized anti-CEA antibody for the treatment of colorectal cancer; bevacizumab (AVASTIN®) which is a humanized anti-VEGF-A mAb for the treatment of brain, colon, kidney or lung cancer; Ibritumomab tiuxetan (ZEVALIN®) which is an anti-CD20 monoclonal antibody for the treatment of non-Hodgkin's lymphoma; ofatumumab (ARZERRA®) which is an anti-human CD20 monoclonal antibody for the treatment of chronic lymphocytic leukemia; panitumumab (VECTIBIX®) which is a monoclonal anti-human EGFR antibody for the treatment of colon cancer; rituximab (RITUXAN®) which is a chimeric anti-CD20 monoclonal antibody for the treatment of chronic lymphocytic leukemia and non-Hodgkin's lymphoma; tositumomab (BEXXAR®) which is an anti-CD20 monoclonal antibody for the treatment of non-Hodgkin's lymphoma; trastuzumab (HERCEPTIN®) which is an anti-HER2 receptor monoclonal antibody for the treatment of breast and stomach cancer; ipilimumab (YERVOY®) which is a human anti-CTLA4 monoclonal antibody for the treatment of melanoma; gemtuzumab and inotuzumab ozogamycin.
[0521] [0521] In another specific embodiment, L includes antibodies selected from anti-I-13 antibodies, including anti-I-13 antibodies used in the treatment of cancer, for example, anti-IL-13R 2 antibodies.
[0522] [0522] In yet another specific embodiment, L includes antibodies selected from anti-Notch antibodies, including anti-Notch antibodies used in the treatment of cancer.
[0523] [0523] In certain embodiments, antibody L is linked to the linker via a sulfur link or via a sulfur-sulfur link.
[0524] [0524] Another aspect of the invention relates to an antibody drug conjugate comprising any of the compounds mentioned above.
[0525] [0525] Another aspect of the invention relates to an antibody drug conjugate comprising an antibody and any of the compounds mentioned above.
[0526] [0526] In certain embodiments, the present invention relates to any of the aforementioned antibody drug conjugates and definitions thereof, wherein the compound is covalently linked to the antibody.
[0527] [0527] In certain embodiments, the present invention relates to any of the aforementioned antibody drug conjugates and definitions thereof, wherein the compound in said antibody drug conjugate is selected from the group consisting of the compounds in Table 18B , O O H
[0528] [0528], O O H
[0529] [0529], O O H
[0530] [0530] O O O O O, O O H
[0531] [0531] O O O O O, O O O
[0532] [0532] and O O O
[0533] [0533].
[0534] [0534] In certain embodiments, the present invention relates to any of the aforementioned antibody drug conjugates and definitions thereof, wherein the antibody drug conjugate comprises between 2, 3, 4, 5, 6, 7, 8, 9 or 10 compounds of the invention.
[0535] [0535] In certain embodiments, the present invention relates to any of the aforementioned antibody drug conjugates and definitions thereof, wherein the antibody drug conjugate comprises 3 or 4 compounds of the invention.
[0536] [0536] The Antibody Unit (Ab)
[0537] [0537] As noted above, the term "antibody" (or "Ab"), which is used here in the broadest sense and specifically covers intact monoclonal antibodies,
[0538] [0538] Heteroatoms that may be present in an antibody unit include sulfur (in one embodiment, from a sulfhydryl group of an antibody), oxygen (in one embodiment, from a carbonyl, carboxyl or hydroxyl group in one antibody) and nitrogen (in one embodiment, from a primary or secondary amino group of an antibody). These heteroatoms can be present in the antibody in the natural state of the antibody, for example, a naturally occurring antibody, or can be introduced into the antibody through chemical modification.
[0539] [0539] In one embodiment, an antibody unit has a sulfhydryl group and the antibody unit bonds through the sulfur atom of the sulfhydryl group.
[0540] [0540] In another embodiment, the antibody has lysine residues that can react with activated esters (such esters include, but are not limited to, N-hydroxysuccinimide, pentafluorophenyl, and p-nitrophenyl esters) and, thus, forms a amide bond consisting of a nitrogen atom of the antibody unit and a carbonyl.
[0541] [0541] In yet another aspect, the antibody unit has one or more lysine residues that can be chemically modified to introduce one or more sulfhydryl groups. Reagents that can be used to modify lysines include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and 2-iminothiolane hydrochloride (Traut's reagent).
[0542] [0542] In another embodiment, the antibody unit can have one or more groups of carbohydrates that can be chemically modified to have one or more sulfhydryl groups.
[0543] [0543] In yet another embodiment, the antibody unit can have one or more carbohydrate groups that can be oxidized to provide an aldehyde group (see, for example, Laguzza et al., 1989, J. Med. Chem. 32 ( 3): 548-55). The corresponding aldehyde can form a bond with a reactive site, such as, for example, hydrazine and hydroxylamine. Other protocols for modifying proteins for binding or associating drugs are described in Coligan et al., Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002) (incorporated herein by reference).
[0544] [0544] When the conjugates comprise non-immunoreactive protein, polypeptide, or peptide units instead of an antibody, useful non-immunoreactive proteins, polypeptide, or peptide units include, but are not limited to, transferrin, epidermal growth factors ("EGF"), bombesin,
[0545] [0545] Useful polyclonal antibodies are heterogeneous populations of antibody molecules derived from the serum of immunized animals. Useful monoclonal antibodies are homogeneous populations of antibodies to a particular antigenic determinant (for example, a cancer cell antigen, a viral antigen, a microbial antigen, a protein, a peptide, a carbohydrate, a chemical, nucleic acid, or fragments of the same). A monoclonal antibody (mAb) of an antigen of interest can be prepared using any technique known in the art, which provides for the production of antibody molecules by continuous cell lines in culture.
[0546] [0546] Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, antibody fragments, or chimeric monoclonal antibodies. Human monoclonal antibodies can be produced by any of the numerous techniques known in the art (for example, Teng et al., 1983, Proc. Natl. Acad. Sci. USA.
[0547] [0547] The antibody can also be a bi-specific antibody. Methods for producing bispecific antibodies are known in the art and are discussed below.
[0548] [0548] The antibody can be a functionally active fragment, a derivative or analogue of an antibody that immunospecifically binds to target cells (eg cancer cell antigens, viral antigens, or microbial antigens), or other antibodies that bind to tumor cells or matrix. In this regard, "functionally active" means that the fragment, derivative or analogue is capable of inducing anti-anti-idiotype antibodies that recognize the same antigen as the antibody from which the recognized fragment, derivative or analog is derived. Specifically, in an exemplary embodiment, the antigenicity of the immunoglobulin molecule idiotype can be increased by removing structures and CDR sequences that are at the C-terminus for the CDR sequence that specifically recognizes the antigen. To determine which CDR sequences bind to the antigen, synthetic peptides containing the CDR sequences can be used in agglutination assays with the antigen by any agglutination assay method known in the art (for example, the BIA core assay) (for the location of CDR sequences, see, for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md .; Kabat E et al., 1980, J .
[0549] [0549] Other useful antibodies include antibody fragments such as, but not limited to, F (ab ') 2, Fab fragments, Fvs, single chain antibodies, diabody, tribody, tetribody, scFv, scFv-FV, or any another molecule with the same specificity as the antibody.
[0550] [0550] Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both non-human and human portions, which can be made using standard recombinant DNA techniques, are useful antibodies. A chimeric antibody is a molecule, in which different portions are derived from different animal species, such as, for example, those that have a variable region derived from a murine monoclonal and constant regions of human immunoglobulin. (See, for example, U.S. Patent No. 4,816,567; and U.S. Patent No. 4,816,397, which are incorporated herein by reference in their entirety). Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from non-human species and a structure region from a human immunoglobulin molecule. (See, for example, U.S. Patent No. 5,585,089, which is incorporated herein by reference in its entirety). Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example, using the methods described in International Publication No. WO 87/02671; European Patent Publication No. 0 184 187; European Patent Publication No. 0 171 496; European Patent Publication No. 0 173 494; International Publication No. WO 86/01533; U.S. Patent No. 4,816,567; European Patent Publication No. 012 023; Berter et al., 1988, Science 240: 1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84: 3439-3443; Liu et al., 1987, J. Immunol. 139: 3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84: 214-218; Nishimura et al., 1987, Cancer. Res. 47: 999-1005; Wood et al., 1985, Nature 314: 446-449; and Shaw et al., 1988, J. Natl. Cancer Inst. 80: 1553-1559; Morrison, 1985, Science 229: 1202-1207; Oi et al., 1986, BioTechniques 4: 214; U.S. Patent No. 5,225,539; Jones et al., 1986, Nature 321: 552-525; Verhoeyan et al., 1988, Science 239: 1534; and Beidler et al., 1988, J. Immunol. 141: 4053-4060; each of which is incorporated herein by reference in its entirety.
[0551] [0551] Completely human antibodies are particularly desirable and can be produced using transgenic mice that are unable to express endogenous immunoglobulin heavy and light chain genes, but which can express human heavy and light chain genes. Transgenic mice are immunized in the normal way with a selected antigen, for example, all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. Human immunoglobulin transgenes harbored by transgenic mice reorganize during B cell differentiation and subsequently undergo class change and somatic mutation. Thus, using this technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for the production of human antibodies, see Lonberg and Huszar, 1995, Int. Rev. Immunol. 13: 65-93. For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, for example, U.S. Patent Numbers 5,625,126;
[0552] [0552] Completely human antibodies that recognize a selected epitope can be generated using a technique known as "guided selection". In this approach, a selected non-human monoclonal antibody, for example, a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (See, for example, Jespers et al., 1994, Biotechnology 12: 899-903). Human antibodies can also be produced using various techniques known in the art, including phage display libraries (see, for example, Hoogenboom and Winter, 1991, J. Mol. Biol. 227: 381; Marks et al., 1991, J Mol. Biol. 222: 581; Quan and Carter, 2002, The rise of monoclonal antibodies as therapeutics, In anti-IgE and Allergic Disease, Jardieu and Fick, eds., Marcel Dekker, New York, NY, Chapter 20, pp. 427- 469).
[0553] [0553] In other embodiments, the antibody is an antibody fusion protein, or a functionally active fragment thereof, for example, in which the antibody is fused via a covalent bond (for example, a peptide bond) , either the N-terminal or the C-terminal to an amino acid sequence of another protein (or portion thereof, preferably at least 10, 20 or 50 amino acid portion of the protein) that is not of an antibody. Preferably, the antibody or fragment thereof is covalently linked to the other protein at the N-terminal of the constant domain.
[0554] [0554] Antibodies include analogs and derivatives that are modified, that is, through covalent bonding of any type of molecule, just as such covalent bonding allows the antibody to retain its antigen agglutination immunospecificity. For example, but not by way of limitation, derivatives and analogs of antibodies include those that have been further modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, binding to a cell antibody unit or other protein, etc. Any of the numerous chemical modifications can be carried out by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic syntheses in the presence of tunicamycin, etc. In addition, the analog or derivative may contain one or more unnatural amino acids.
[0555] [0555] Antibodies may have modifications (for example, substitutions, deletions or additions) in the amino acid residues that interact with Fc receptors. In particular, antibodies may have modifications to amino acid residues identified as involved in the interaction between the anti-Fc domain and the FcRn receptor (see, for example, International Publication No. WO 97/34631, which is incorporated herein by reference. in your totality).
[0556] [0556] Immuno-specific antibodies to a cancer cell antigen can be obtained commercially or produced by any method known to one skilled in the art, such as, for example, chemical syntheses or recombinant expression techniques. The nucleotide sequence encoding the immunospecific antibodies to a cancer cell antigen can be obtained, for example, from the GenBank database or a similar database, literature publications, or by cloning and sequencing routine.
[0557] [0557] In a specific embodiment, antibodies known to treat cancer can be used. Immuno-specific antibodies to a cancer cell antigen can be obtained commercially or produced by any method known to one skilled in the art, such as, for example, recombinant expression techniques. The nucleotide sequence encoding the immunospecific antibodies to a cancer cell antigen can be obtained, for example, from the GenBank database, or a database like the same, literature publications, or by routine cloning and sequencing.
[0558] [0558] In another specific embodiment, anti-IL13 antibodies, including anti-IL13 antibodies used in the treatment of cancer, can be used.
[0559] [0559] In another specific embodiment, anti-Notch antibodies, including anti-Notch antibodies used in the treatment of cancer, can be used.
[0560] [0560] In attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers sought to identify the transmembrane, or otherwise, tumor-associated polypeptides that are specifically expressed on the surface of one or more particular types of cancer cell compared to one or more normal non-cancer cells. Often, these tumor-associated polypeptides are more abundantly expressed on the surface of cancer cells compared to the surface of non-cancer cells. The identification of such tumor-associated cell surface antigen polypeptides has provided the possibility for specifically targeted cancer cells through antibody-based therapies.
[0561] [0561] Synthesis of Antibody Drug Compounds and Conjugates
[0562] [0562] The compounds and conjugates of the present invention can be made using the synthetic procedures described below in the Exemplification.
[0563] [0563] As described in more detail below, the compounds and conjugates of the present invention can be prepared using a section of a linker unit having a reactive site for agglutination to the compound.
[0564] [0564] Binder
[0565] [0565] A linker (sometimes referred to as "[linker]" here) is a bifunctional compound that can be used to link a drug and an antibody to form an antibody drug conjugate (ADC). These conjugates are useful, for example, in the formation of immunoconjugates directed against antigens associated with the tumor. Such conjugates allow for selective delivery of cytotoxic drugs to tumor cells.
[0566] [0566] In one embodiment, the linker has the formula:
[0567] [0567] is or where:
[0568] [0568] Y is C2-C20 alkylene or C2-C20 heteroalkylene; C3-C8 carbocyclo-, -arylene-, C3-C8 heterocyclo-, -C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene, - C1-C10 alkylene- (C3-C8 carbocycle) -, - (C3 -C8 carbocycle) - C1-C10 alkylene-, -C1-C10 alkylene- (C3-C8 heterocycle) -, or - (C3-C8 heterocycle) -C1-C10 alkylene-; Zé, O H N H O N
[0569] [0569] R7 is selected, independently, for each occurrence from the group consisting of F, Cl, I, Br, NO2, CN and CF3;
[0570] [0570] R10 is hydrogen, -C1-C10 alkyl, -C3-C8 carbocycle, aryl, -C1-C10 heteroalkyl, - C3-C8 heterocycle, -C1-C10 alkylene-aryl, -arylene-C1-C10 alkyl, - C1-C10 alkylene- (C3-C8 carbocycle), - (C3-C8 carbocycle) -C1-C10 alkyl, -C1-C10 alkylene- (C3-C8 heterocycle), and - (C3-C8 heterocycle) -C1-C10 alkyl, where aryl at R10 which comprises aryl is optionally substituted with [R7] h; and
[0571] [0571] h is 1, 2, 3, 4 or 5.
[0572] [0572] In an ADC, the ligand serves to fix the charge for the antibody.
[0573] [0573] In one aspect, a second section of the linker unit is introduced, which has a second reactive site, for example, an electrophilic group that is reactive to a nucleophilic group present in an antibody unit (for example, an antibody) . Nucleophilic groups useful on an antibody include, but are not limited to, sulfhydryl, hydroxyl and amino groups. The heteroatom of the nucleophilic group of an antibody is reactive to an electrophilic group for a linker and forms a covalent bond with a linker. Useful electrophilic groups include, but are not limited to, maleimide and haloacetamide groups. The electrophilic group provides a convenient location for antibody binding.
[0574] [0574] In another embodiment, a linker unit has a reactive site that has a nucleophilic group that is reactive to an electrophilic group present in an antibody.
[0575] [0575] Functional amino groups are also useful reactive sites for a linker unit, because they can react with the carboxylic acid, or activated esters of a compound, to form an amide bond.
[0576] [0576] As described in more detail below, conjugates can be prepared using a section of the linker having a reactive site for agglutination to a compound of the invention and introducing another section of the linker unit having a reactive site for an antibody. In one aspect, a linker unit has a reactive site that has an electrophilic group that is reactive with a nucleophilic group present in an antibody unit, such as an antibody. The electrophilic group provides a convenient location for antibody binding.
[0577] [0577] In another embodiment, a linker unit has a reactive site that has a nucleophilic group that is reactive with an electrophilic group present in an antibody unit. The electrophilic group in an antibody provides a convenient location for binding to a binding unit. Electrophilic groups useful on an antibody include, but are not limited to, aldehyde and carbonyl ketone groups. The heteroatom of a nucleophilic group of a linker can react with an electrophilic group in an antibody and form a covalent bond to the antibody. Useful nucleophilic groups on a linker include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate and arylhydrazide.
[0578] [0578] As used here, "mc-" formerly known as "MalC-" refers to.
[0579] [0579] As used here, "mcValCitPABC-" formerly known as "MalCValCitPABC-" refers to. O O N O X
[0580] [0580] As used here, "MalPegXC2-" refers to O.
[0581] [0581] As used herein, "AmPegXC2-" refers to X.
[0582] [0582] As used here, "mcValCitPABCAmPegXC2-" refers to O O O O O O N H H X N N O N N The H H O
[0583] [0583] As used here, "MalPegXC2ValCitPABC-" refers to O O O O O H N N The X N N The H H O
[0584] [0584] As used herein, "2BrAcPegXC2" refers to O. O N
[0585] [0585] As used here, "mv-" refers to O O. O O N
[0586] [0586] As used here, "mb-" refers to O. O N
[0587] [0587] As used here, "me-" refers to O O. O N
[0588] [0588] As used here, "MalC6-" refers to O.
[0589] [0589] As used here, "PFPCOPegXC2ValCitPABC-" refers to F O F F O O O O H The N F O X N N H H F O
[0590] [0590] As used here, "PFPCOPegXC2AmPegYC2-" refers to F F F O O H N F O O O X X F O.
[0591] [0591] As used here, "PFPCOPegXC2AlaAlaAsnPABC-" refers to F NH2 F F O O O O H H F O O N N N X N H H F O O O O. F F F O O F O O X
[0592] [0592] As used herein, "PFPCOPegXC2-" refers to F.
[0593] [0593] As used here, "PFPCOPegXC2AmPegYC2PABC-" refers to F F F O O O H AT THE F O O O N X X H F O. O O N N H
[0594] [0594] As used here, "mcGly-" refers to O O.
[0595] [0595] As used here, "AzCOC2Ph4AmPeg1C1-" refers to O O O H H N N The N N H H N O O O
[0596] [0596] As used here, "AcLysValCitPABC-" refers to.
[0597] [0597] Conjugation with Transglutaminase
[0598] [0598] In certain embodiments, a compound of the invention can be covalently cross-linked to an Fc-containing or Fab-containing polypeptide constructed with a label containing acyl donor glutamine (e.g.
[0599] [0599] Conjugation to the Human Light Chain Kappa Domain Constant Region
[0600] [0600] In certain embodiments, a compound of the invention can be covalently linked to the K188 side chain of the human light chain (CL) kappa domain constant region (complete light chain numbering according to Kabat).
[0601] [0601] For example, the compounds of the invention can be conjugated, as described in U.S. Patent Application Serial Number 13 / 180,204, or WO2012 / 007896 the entire content of which is incorporated herein by reference. In certain embodiments, to facilitate conjugation for K188 CL (CL -K80), Z is; R7 is selected, independently, for each occurrence from the group consisting of F, Cl, I, Br, NO2, CN and CF3; and h is 1, 2, 3, 4 or 5.
[0602] [0602] In certain modalities, to facilitate the conjugation of K188 CL (CL -K80), Z is;
[0603] [0603] The present invention further provides antibody drug conjugates comprising an antibody, or antigen agglutination portions thereof, comprising a constant kappa domain covalently conjugated to a toxin of the invention, characterized by the fact that at least one toxin of the invention is covalently conjugated to K80 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 (Table 1). In some respects, the number of toxins of the invention covalently conjugated to K80 may be a range, the lower limit of which is selected from the group consisting of about 1.5, about 1.6, about 1.7 , about 1.8, about 1.9, and about 2.0, and whose upper limit is selected from the group consisting of about 2.0, about 2.1, about 2.2 , about 2.3, about 2.4, about 2.5. In some ways, p is about 2.
[0604] [0604] Conjugation of the toxin with the constant light domain of an antibody is particularly desirable to minimize or prevent any interference with the agglutination of the Fc portion of the antibody to Fc receptors (such as. Fc R and FcRn) or agglutination of the antibody to their respective target. Conversely, conjugation of the respective toxin to the Fe portion of an antibody can reduce the antibody's half-life in vivo and / or its ability to interact with the immune system (effector function). The conjugation of the toxin in the variable heavy chain (VH) or variable light chain (VL) region of the antibody carries a risk of decreasing the agglutination of the antibody to its cognate.
[0605] [0605] Furthermore, while the CL-K80 conjugation is reliable and robust to other antibody surface lysines, each of the slightly different reactivity and pI may result in a heterogeneous sample of conjugated antibodies that can distribute the conjugated molecules at inopportune or irregular moments, such as during circulation and before the distribution of the Effective Portion to the target by antibody recognition.
[0606] [0606] In addition, the present invention provides for known kappa V / A polymorphisms at position 45 and A / L at position 83 (providing the 3 human constant kappa polymorphisms identified Km (1): V45 / L83 (SEQ ID NO : 2), Km (1,2): A45 / L83 (SEQ ID NO: 3), and Km (3) A45 / V83 (SEQ ID NO: 4)). The variability of residues at positions 45 and 83 in SEQ ID NO: 1 can be selected so as to provide only for any one, two or all three polymorphisms Km (1), Km (1,2), and Km (3 ).
[0607] [0607] where X at position 45 is A or V, and X at position 83 is L or V.
[0608] [0608] In certain embodiments, the invention provides a composition comprising a compound of the invention covalently conjugated to an antibody (or antigen agglutination portion thereof), wherein at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the compound of the invention, wherein the composition is conjugated to the antibody or antigen agglutination portion thereof in K188 CL.
[0609] [0609] In certain embodiments, the compounds of the invention,,
[0610] [0610] and can be conjugated to the combination site of a catalytic antibody, such as aldolase antibodies, or antigen agglutination portion thereof. Aldolase antibodies contain antibodies, portions of the combination site that, when free (for example, by conjugation), catalyze an aldol addition reaction between an aliphatic ketone donor and an aldehyde acceptor. The contents of US Patent Application Publication No. US 2006/205670 are incorporated herein by reference, in particular, pages 78-118 describe the ligands, and paragraphs [0153] - [0233] describe the antibodies, useful fragments, variants and modifications thereof, h38C2, combination sites and complementary determinant regions (CDRs), and related antibody technology (Table 2, and exemplary compounds below): O H O H O H N N N N The OH The N N O O O O O O N
[0611] [0611] O O, N O O H H N N N S O O N N H H H O O N N O O O The N N H H N O O O The NH
[0612] [0612] H2N O, N O O H H N N N S O O N N H H H O O N N O O O The N N H H N O O O The NH
[0613] [0613] H 2N O e N H O O H H N N O N N S The N N H O O N O O O
[0614] [0614] O O
[0615] [0615] The term "combination site" includes CDRs and adjacent structure residues that are involved in antigen agglutination.
[0616] [0616] Conjugation with binders comprising succinimides, including open ring versions
[0617] [0617] In certain embodiments, the present invention includes a compound of the invention conjugated via a succinimide-based linker or an open ring succinimide-based linker. The stability of the succinimide-cysteine bond has become an area of increasing interest. Succinimides can be transferred both in vitro and in vivo to exogenous thiol nucleophiles, presumably through a retro-Michael reaction resulting in a maleimide that is subsequently attacked by a thiol. It is believed that hydrolysis of the ring results in a species that is resistant to the retro-Michael reaction. This makes the resulting conjugate more stable and potentially more effective. Conditions can be optimized to forcibly open the succinimide ring in the conjugate. Basic conditions resulted in easy hydrolysis of the ring.
[0618] [0618] Example of forced hydrolysis of maleimide-based conjugates
[0619] [0619] In order to assess the stability of these conjugates and give priority to samples for in vivo evaluation, an assay has been developed that involves the treatment of conjugates bound to maleimide with excess aqueous glutathione (GSH) or plasma. Aliquots of the reaction mixture were analyzed at various time points to determine the conjugate loading, using the methodology described above. The results (Table 24) show that the drug-antibody binding is slowly cleaved in a GSH-dependent manner. As expected, the rate of cleavage is highly dependent on the hydrolysis of the succinimide ring. More importantly, these results seem to translate the improved PK exposure, as measured by an increase in the area under the curve (AUC) of the conjugate and an increase in the conjugate / Ab exposure rate.
[0620] [0620] Method for assessing the stability of ADCs
[0621] [0621] The ADC sample (30 µg) in PBS is mixed with a solution of glutathione (GSH) to produce a final concentration of 0.5 mM GSH and 3 mg / mL protein concentration. A control sample (without GSH) was also prepared from 30 µg of ADC diluted to 3 mg / mL in PBS. The GSH-treated ADC sample and control ADC sample were incubated at 37 ° C and were sampled at 0, 3, and 6 days. The aliquots were reduced with excess TCEP, acidified by adding 0.1% formic acid solution with 10% acetonitrile and analyzed for LC / MS loading as described below.
[0622] [0622] Sample analysis: The analyzes were performed using an Aglient 1100 capillary HPLC coupled with a Waters Xevo G2 Q-TOF mass spectrometer. The analytes were loaded onto a Zorbax Poroshell 300SB C8 column (0.5 mm x 75 mm maintained at 80 ° C) with 0.1% formic acid, and eluted using a 20-40% gradient of buffer B (80% acetonitrile , 18% 1-propanol, 2% water with 0.1% formic acid), at a flow rate of 20 µl / min for 5.5 minutes. Detection by mass spectrometry was performed in the positive mode, sensitivity with the capillary voltage set at 3.3 kV. Data analysis was performed with the MaxEnt 1 function in MassLynx and intensities were used to calculate the load based on the formula described above.
[0623] [0623] Compositions and Methods of Administration
[0624] [0624] In other embodiments, another aspect of the invention relates to pharmaceutical compositions including an effective amount of a compound of the invention and / or the antibody drug conjugate thereof, and a pharmaceutically acceptable carrier or vehicle. In certain embodiments, the compositions are suitable for human or veterinary administration.
[0625] [0625] The present pharmaceutical compositions can be in any form that allows the composition to be administered to a patient. For example, the composition can be in the form of a liquid or a solid. Typical routes of administration include, without limitation, parenteral, ocular and intratumor. Parenteral administration includes subcutaneous injections, intravenous, intramuscular or intrasternal injection or infusion techniques. In one aspect, the compositions are administered parenterally. In a specific embodiment, the compositions are administered intravenously.
[0626] [0626] The pharmaceutical compositions can be formulated so as to allow a compound of the invention and / or the antibody drug conjugate thereof to be bioavailable by administering the composition to a patient. The compositions can take the form of one or more dosage units, wherein, for example, a tablet can be a single dosage unit, and a container of a compound of the invention and / or the antibody drug conjugate thereof under the liquid form can contain a plurality of dosage units.
[0627] [0627] The materials used in the preparation of pharmaceutical compositions can be non-toxic in the amounts used. It will be apparent to those skilled in the art that the optimal dose of active ingredients in the pharmaceutical composition will depend on a variety of factors. Relevant factors include, without limitation, the type of animal (e.g., human), the particular form of a compound of the invention and / or the antibody drug conjugate thereof, the mode of administration, and the composition employed.
[0628] [0628] The pharmaceutically acceptable carrier or vehicle can be solid or particulate, so that the compositions are, for example, in tablet or powder form. Vehicles can be liquid. In addition, vehicles can be particulate.
[0629] [0629] The composition can be in the form of a liquid, for example, a solution, emulsion, or suspension. In a composition for administration by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included.
[0630] [0630] Liquid compositions, whether they are solutions, suspensions or the like, can also include one or more of the following: sterile diluents, such as water for injection, saline, preferably physiological saline, Ringer's solution, chloride isotonic sodium, fixed oils, such as mono synthetics or diglycerides that can serve as the solvent or suspending medium, polyethylene glycols, glycerin, cyclodextrin, propylene glycol or other solvents; antibacterial agents, such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylene diaminetetraacetic acid; buffers, such as acetates, citrates, phosphates or amino acids and agents for adjusting tonicity, such as sodium chloride or dextrose. A parenteral composition can be closed in ampoules, a disposable syringe or a multiple dose vial made of glass, plastic or other material. Physiological saline is an exemplary adjuvant. An injectable composition is preferably sterile.
[0631] [0631] The amount of a compound of the invention and / or the antibody drug conjugate thereof, which is effective in treating a particular disorder or condition, will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques . In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be used in the compositions will also depend on the route of administration, and the severity of the disease or disorder, and should be decided according to the judgment of the physician and the circumstances of each patient.
[0632] [0632] The compositions comprise an effective amount of a compound of the invention and / or the antibody drug conjugate thereof, such that a suitable dosage will be obtained. Typically, this amount is at least about 0.01% of a compound of the invention and / or the antibody drug conjugate of the same, by weight, of the composition. In an exemplary embodiment, pharmaceutical compositions are prepared so that a parenteral dosage unit contains between about 0.01% to about 2%, by weight, of the amount of a compound of the invention and / or the drug conjugate of antibody to it.
[0633] [0633] For intravenous administration, the composition can comprise from about 0.01 to about 100 mg of a compound of the invention and / or the antibody drug conjugate thereof per kg of body weight of the patient. In one aspect, the composition can include from about 1 to about 100 mg of a compound of the invention and / or the antibody drug conjugate thereof per kg of patient's body weight. In another aspect, the amount administered will be in the range of about 0.1 to about 25 mg / kg of body weight of a compound of the invention and / or the antibody drug conjugate thereof.
[0634] [0634] Generally, the dosage of a compound of the invention and / or the antibody drug conjugate thereof administered to a patient is typically from about 0.01 mg / kg to about 20 mg / kg of the patient's body weight . In one aspect, the dosage administered to a patient is from about 0.01 mg / kg to about 10 mg / kg of the patient's body weight. In another aspect, the dosage administered to a patient is between about 0.1 mg / kg and about 10 mg / kg of the patient's body weight. In yet another aspect, the dosage administered to a patient is between about 0.1 mg / kg and about 5 mg / kg of the patient's body weight. In yet another aspect, the dosage administered is between about 0.1 mg / kg to about 3 mg / kg of the patient's body weight. In yet another aspect, the dosage administered is from about 1 mg / kg to about 3 mg / kg of the patient's body weight.
[0635] [0635] A compound of the invention and / or the antibody drug conjugate thereof can be administered by any convenient route, for example by infusion or bolus injection. Administration can be systemic or local. Various delivery systems are known, for example, encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer a compound of the invention and / or the antibody drug conjugate thereof. In certain embodiments, more than one compound of the invention and / or the antibody drug conjugate thereof is administered to a patient.
[0636] [0636] In specific embodiments, it may be desirable to administer one or more compounds of the invention and / or the antibody drug conjugates thereof locally to the area in need of treatment. This can be achieved, for example, and not by way of limitation, by local infusion during surgery; topical application, for example, in conjunction with a wound dressing after surgery; by injection; through a catheter; or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including membranes, such as silastic membranes, or fibers. In one embodiment, administration can be by direct injection into the site (or old site) of cancer, tumor, or neoplastic or pre-neoplastic tissue. In another embodiment, administration can be by direct injection into the site (or old site) of an autoimmune disease manifestation.
[0637] [0637] In yet another embodiment, the compound of the invention and / or the antibody drug conjugate thereof can be delivered in a controlled release system, such as, but not limited to, a pump or various polymeric materials can be used . In yet another embodiment, a controlled release system can be placed in close proximity to the target of the compound of the invention and / or the antibody drug conjugate thereof, for example, the liver, thus requiring only a fraction of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled-release systems discussed in the journal by Langer (Science 249: 1527-1533 (1990)) can be used.
[0638] [0638] The term "carrier" refers to a diluent, adjuvant or excipient, with which a compound or antibody drug conjugate thereof is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. The carriers can be serum and the like. In addition, auxiliary, stabilizing and other agents can be used. In one embodiment, when administered to a patient, the pharmaceutically acceptable compounds or conjugates and carriers are sterile. Water is an exemplary carrier when the compound or conjugate is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
[0639] [0639] The present compositions can take the form of solutions, pellets, powders, controlled release formulations, or any other form suitable for use.
[0640] [0640] In one embodiment, the compound of the invention and / or the antibody drug conjugate thereof is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to animals, particularly humans. Typically, carriers or vehicles for intravenous administration are sterile isotonic aqueous buffer solutions. Where necessary, the compositions can also include a solubilizing agent. Compositions for intravenous administration can optionally include a local anesthetic, such as lignocaine to relieve pain at the injection site. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry or water-free concentrated lyophilized powder in a hermetically sealed container, such as an ampoule or sachet indicating the amount of active agent.
[0641] [0641] The composition may include various materials that modify the physical form of a solid or liquid dosage unit. For example, the composition can include materials that form a coating wrap around the active ingredients. The materials that form the coating shell are usually inert, and can be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients can be incorporated into a gelatin capsule.
[0642] [0642] Whether in solid or liquid form, the present compositions can include a pharmacological agent used in the treatment of cancer.
[0643] [0643] Therapeutic Uses of Antibody Drug Compounds and Conjugates
[0644] [0644] Another aspect of the invention relates to a method of using the compounds of the invention and antibody drug conjugates thereof to treat cancer.
[0645] [0645] The compounds of the invention and / or the antibody drug conjugates thereof, are useful for inhibiting the multiplication of a tumor cell or cancer cell, causing apoptosis in a tumor or cancer cell, or for treating the cancer in a patient. The compounds of the invention and / or the antibody drug conjugates thereof, can be used according to a variety of configurations for the treatment of cancers in animals. Said conjugates can be used to deliver a compound of the invention to a tumor cell or cancer cell. Without being limited by theory, in one embodiment, the conjugate antibody binds to, or associates with, a cancer cell or an antigen associated with tumor cells, and the conjugate can be made up to (internalized) within a cell. tumor or cancer cells through receptor-mediated endocytosis or other internalization mechanism. The antigen can be attached to a tumor cell or cancer cell, or it can be an extracellular matrix protein associated with the tumor cell or cancer cell. In certain embodiments, once inside the cell, one or more specific peptide sequences are enzymatically or hydrolytically cleaved by one or more tumor cells or proteases associated with cancer cells, resulting in the release of a compound of the invention from the conjugate . The compound released from the invention is then free to migrate within the cell and induce cytotoxic or cytostatic activities. The conjugate can also be cleaved by an intracellular protease to deliver a compound of the invention. In an alternative embodiment, the compound of the invention is cleaved from the conjugate outside the tumor cell or cancer cell, and the compound of the invention subsequently penetrates the cell.
[0646] [0646] In certain embodiments, the conjugates provide a specific conjugate tumor or targeted cancer drug, thereby reducing the general toxicity of the compounds of the invention.
[0647] [0647] In another embodiment, the antibody unit binds to the tumor cell or cancer cell.
[0648] [0648] In another embodiment, the antibody unit binds to a tumor cell or cancer cell antigen, which is on the surface of the tumor cell or cancer cell.
[0649] [0649] In another embodiment, the antibody unit binds to a tumor cell or cancer cell antigen, which is an extracellular matrix protein associated with the tumor cell or cancer cell.
[0650] [0650] The specificity of the antibody unit for a particular tumor cell or cancer cell can be important in determining those tumors or cancers that are treated most effectively.
[0651] [0651] Particular types of cancers that can be treated with a compound of the invention and / or an antibody drug conjugate thereof, include, but are not limited to, bladder carcinoma, breast, cervix, colon, endometrium, kidney , lung, esophagus, ovary, prostate, pancreas, skin, stomach, and testicles; and cancers through the blood, including but not limited to, leukemias and lymphomas.
[0652] [0652] Multimodality Therapy for Cancer. Cancers, including, but not limited to, a tumor, metastasis, or other disease or disorder characterized by uncontrolled cell growth, can be treated or inhibited by administration of a compound of the invention and / or the antibody drug conjugate thereof.
[0653] [0653] In other embodiments, methods for treating cancer are provided, including administering to a patient in need of it, an effective amount of a compound of the invention and / or the antibody drug conjugate thereof and an agent chemotherapeutic. In one embodiment, the chemotherapeutic agent is one with which cancer treatment has not been found to be refractory. In another modality, the chemotherapeutic agent is one with which the cancer treatment was found to be refractory. A compound of the invention and / or the antibody drug conjugate thereof can be administered to a patient who has undergone surgery as a treatment for cancer.
[0654] [0654] In some modalities, the patient also receives additional treatment, such as radiation therapy. In a specific embodiment, the compound of the invention and / or the antibody drug conjugate thereof is administered concomitantly with the chemotherapeutic agent or with radiation therapy. In another specific embodiment, the chemotherapeutic agent or radiation therapy is administered before or after administration of a compound of the invention and / or the antibody drug conjugate thereof.
[0655] [0655] A chemotherapeutic agent can be administered over a series of sessions. Any one or a combination of the chemotherapeutic agents, such as a careful chemotherapeutic agent pattern, can be administered.
[0656] [0656] In addition, methods of treating cancer with a compound of the invention and / or the antibody drug conjugate thereof are provided as an alternative to chemotherapy or radiation therapy where chemotherapy or radiation therapy has proven or can proving to be very toxic, for example, results in unacceptable or intolerable side effects for the individual being treated. The patient to be treated can optionally be treated with another cancer treatment, such as surgery, radiation therapy or chemotherapy, depending on which treatment is considered to be acceptable or tolerable.
[0657] [0657] The compounds of the invention and / or antibody drug conjugates thereof can also be used in an in vitro or ex vivo assay, such as for the treatment of certain types of cancer, including, but not limited to, leukemias and lymphomas, such treatment involves autologous stem cell transplants. This may involve a multi-step process, in which the animal's autologous hematopoietic stein cells were harvested and purged from all cancer cells, the remaining bone marrow cell population of the animal is then eliminated by administering a high dose of a compound of the invention and / or the antibody drug conjugate thereof with or without the accompaniment of high dose radiation therapy, and the stem cell graft is administered back to the animal. Supportive treatment is then provided while bone marrow function is restored and the patient recovers.
[0658] [0658] Released Species
[0659] [0659] Other embodiments of the invention include the chemical species released, inside or on the periphery of the cancer cell or tumor cell with what is believed to be the enzymatic and / or hydrolytic cleavage of one or more cancer cells or proteases associated with tumor cells. Such compounds include the species described herein, and also include compounds, such as those described in the structure: H O H
[0660] [0660] or a pharmaceutically acceptable salt or solvate thereof, where, independently at each occurrence, 1-2 R3A
[0661] [0661] R is.
[0662] [0662] Y is C2-C20 alkylene or C2-C20 heteroalkylene; C3-C8 carbocyclo-, -arylene-, -C3-C8 heterocyclo-, -C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, -C1-C10 alkylene- (C3-C8 carbocycle) -, - (C3-C8 carbocycle) -C1-C10 alkylene-, -C1-C10alkylene- (C3-C8 heterocycle) -, or - (C3-C8 heterocycle) -C1-C10 alkylene-; O O
[0663] [0663] G is halogen, - OH, - SH or -S-C1-C6 alkyl;
[0664] [0664] R2 is hydrogen, C1-C8 alkyl or C1-C6 haloalkyl;
[0665] [0665] R3A and R3B are defined as one of the following: (i) R3A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1- C10 heterocyclyl, aryl, heteroalkyl, aralkyl or halogen; and
[0666] [0666] R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl; or
[0667] [0667] (ii) R3A and R3B, considered together are C2-C8 alkylene or C1-C8 heteroalkylene;
[0668] [0668] R4A and R4B are defined as one of the following: (i) R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroalkyl or aralkyl; and
[0669] [0669] R4B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or
[0670] [0670] (ii) R4A and R4B, considered together are C2-C8 alkylene or C1-C8 heteroalkylene;
[0671] [0671] R5 is,,,
[0672] [0672],,,,
[0673] [0673],,,,
[0674] [0674],,, C1-C10 heterocyclyl, C3-C8 carbocyclyl and C6-C14 aryl optionally substituted with 1, 2, 3, 4 or groups independently selected from the group consisting of -C1-C8 alkyl, -C1- C8 alkyl-N (R ') 2, -C1-C8 alkyl-C (O) R', -C1-C8 alkyl-C (O) OR ', -O (C1-C8 alkyl), -C (O) R ', -OC (O) R', -C (O) OR ', -C (O) N (R') 2, NHC (O) R ', -S (O) 2R', -S (O ) R ', OH, halogen, -N3, -N (R') 2, -CN, -NHC (= NH) NH2, -NHCONH2, -S (= O) 2R 'and -SR', where each R 'is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl, or two R' can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; O
[0675] [0675] or R5 is, R13, or
[0676] [0676] R13 optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of -C1-C8 alkyl, -C1-C8 alkyl-N (R ') 2, -C1-C8 alkyl -C (O) R ', -C1-C8 alkyl-C (O) OR', -O (C1-C8 alkyl), - C (O) R ', -OC (O) R', -C (O ) OR ', -C (O) N (R') 2, NHC (O) R ', -S (O) 2R', -S (O) R ', OH, halogen, -N3, -N (R ') 2, -CN, -NHC (= NH) NH2, -NHCONH2, -S (= O) 2R' and -SR 'and arylene -R', where each R 'is independently selected from the group consisting of in hydrogen, C1-C8 alkyl, C1-C8 heterocyclyl, -C1-C10 alkylene-C3-C8 heterocyclyl and aryl, or two R 'can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl;
[0677] [0677] R6 is hydrogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -C1-C8 haloalkyl;
[0678] [0678] R12 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl;
[0679] [0679] R13 is C1-C10 heterocyclyl; and
[0680] [0680] R7 is independently selected for each occurrence, from the group consisting of F, Cl, I, Br, NO2, CN and CF3;
[0681] [0681] R10 is hydrogen, -C1-C10 alkyl, -C3-C8 carbocycle, aryl, -C1-C10 heteroalkyl, - C3-C8 heterocycle, -C1-C10 alkylene-aryl, -arylene-C1-C10 alkyl, - C1-C10 alkylene- (C3-C8 carbocycle), - (C3-C8 carbocycle) -C1-C10 alkyl, -C1-C10 alkylene- (C3-C8 heterocycle), and - (C3-C8 heterocycle) -C1-C10 alkyl, where aryl at R10 which comprises aryl is optionally substituted with [R7] h;
[0682] [0682] h is 1, 2, 3, 4 or 5; and
[0683] [0683] X is O or S.
[0684] [0684] Of particular interest are compounds of formula IV, having the structures: The OH H H N N N
[0685] [0685] The invention is further described in the following examples, which are not intended to limit the scope of the invention. EXEMPLIFICATION
[0686] [0686] The experiments were generally carried out under an inert atmosphere (nitrogen or argon), particularly in cases where reagents or intermediates sensitive to moisture or oxygen were used. Commercial solvents and reagents were generally used without further purification, including anhydrous solvents when appropriate (generally, Sure-SealTM products from Aldrich Chemical Company, Milwaukee, Wisconsin). The products were generally dried under vacuum before being transported to other reactions or subjected to biological testing. Mass spectrometry data is reported from any liquid chromatography mass spectrometry (LCMS), atmospheric pressure chemical ionization (APCI) or gas chromatography mass spectrometry instrumentation (GCMS). The chemical deviations for nuclear magnetic resonance (NMR) data are expressed in parts per million (ppm,) referring to residual peaks of the deuterated solvents employed.
[0687] [0687] For syntheses referencing procedures in other Examples or Methods, reaction protocol (reaction length and temperature) may vary.
[0688] [0688] Optical rotations were performed on a Perkin - Elmer 343 polarimeter (serial number 9506).
[0689] [0689] HRMS were performed on an Agilent 6220 TOF LC / MS.
[0690] [0690] Composite names were generated with ACD Labs software.
[0691] [0691] HPLC and LC-MS conditions used for analysis
[0692] [0692] Protocol A: Column: Phenomenex Luna C18 (2), 150 x 3.0 mm, 5 µm; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient: 5% B for 1.5 minutes, 5% to 100% B for 8.5 minutes, then 100% B for 1 minute; Flow rate: 0.75 mL / minute. Temperature: 25 ° C; Detection: DAD at 215 nm, 254 nm; MS (+) ranges from 150-2000 daltons; Injection volume: 10 µL; Instrument: Agilent 1200 LCMS.
[0693] [0693] Protocol B: Column: Phenomenex Luna C18 (2), 150 x 3.0 mm, 5 µm; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient: 50% B for 1.5 minutes, 50% to 100% B for 6.5 minutes, then 100% B for 3 minutes; Flow rate: 0.75 mL / minute. Temperature: 25 ° C; Detection: DAD 215 nm; MS (+) ranges from 150-2000 daltons; Injection volume: 10 µL; Instrument: Agilent 1200 LCMS.
[0694] [0694] Protocol C: Column: Phenomenex Luna C18 (2), 150 x 3.0 mm, 5 µm; Mobile phase A: 0.02% trifluoroacetic acid in water (v / v); Mobile phase B: 0.02% trifluoroacetic acid in methanol (v / v); Gradient: 50% to 100% B over 10 minutes; Flow rate: 0.75 mL / minute. Temperature: uncontrolled; Detection: DAD at 215 nm, 254 nm; Injection volume: 10 µL; Instrument: Agilent 1100 HPLC.
[0695] [0695] Protocol D: Column: Phenomenex Luna C18 (2), 150 x 3.0 mm, 5 µm; Mobile phase A: 0.02% trifluoroacetic acid in water (v / v); Mobile phase B: 0.02% trifluoroacetic acid in methanol (v / v); Gradient: 5% to 100% B over 8 minutes; Flow rate: 0.75 mL / minute. Temperature: uncontrolled; Detection: DAD at 215 nm, 254 nm; Injection volume: 10 µL; Instrument: Agilent 1100 HPLC.
[0696] [0696] Protocol E: Column: Phenomenex Lux Amylose - 2, 250 x 4.6 mm, 5 µm; Mobile phase A: heptane; Mobile phase B: ethanol (denatured); Gradient: 5% to 100% B over 10 minutes; Flow rate: 1.5 mL / minute. Temperature: non-
[0697] [0697] Protocol F: Column: Waters Acquity UPLC BEH C18, 2.1 x 50 mm, 1.7 µm; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient: 5% B for 0.1 minute, 5% to 95% B for 0.7 minute, 95% B for 0.1 minute; Flow Rate: 1.25 mL / minute.
[0698] [0698] Protocol G: Column: Phenomenex Luna C18 (2), 150 x 3.0 mm, 5 µm; Mobile phase A: 0.02% trifluoroacetic acid in water (v / v); Mobile phase B: 0.02% trifluoroacetic acid in acetonitrile (v / v); Gradient: 0% to 100% B over 8.5 minutes; Flow rate: 1.5 mL / minute. Temperature: uncontrolled; Detection: DAD 210 nm; Injection volume: 10 µL; Instrument: Agilent 1100 HPLC.
[0699] [0699] Protocol H: Column: Phenomenex Gemini-NX, C18, 4.6 x 50 mm, 3 µm, 110 Å; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient: 0% to 100% B for 4.10 minutes, linear, then 100% B for 0.4 minutes; Flow Rate: 1.5 mL / minute. Temperature: 60 ° C; Detection: DAD 200-450 nm; MS (+) ranges from 100-2000 daltons; Injection volume: 5 µL; Instrument: Agilent.
[0700] [0700] Protocol I: Column: Atlantis T3, 75 x 3.0 mm, 3 µM; Mobile phase A: 0.05% trifluoroacetic acid in water (v / v); Mobile phase B: acetonitrile; Gradient: 5% to 95% B over 5.75 minutes; Flow Rate: 1.2 mL / minute. Temperature: 45 ° C; Detection: DAD at 215 nm, 230 nm, 254 nm; MS (+) ranges from 150-1200 daltons; Injection volume: 5 µL; Instrument: Agilent 1100 LCMS.
[0701] [0701] Protocol J: Column: Phenomenex Luna Fenil - Hexil, 150 x 3.0 mm, 5 µm; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient: 5% B over 1.5 minutes, 5% to 100%
[0702] [0702] Protocol K: Column: Symmetry - C18, 50 x 2.1 mm, 3.5 µm; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in methanol (v / v); Gradient: 10% to 90% B over 6.5 minutes; Flow rate: 0.7 mL / minute. Temperature: room temperature; Detection: DAD at 215 nm; MS (+) ranges from 100-1500 daltons; Injection volume: 3 µL; Instrument: Waters 996 PDA.
[0703] [0703] Protocol L: Column: XBridge C - 18, 150 x 4.6 mm, 3.5 µm; Mobile phase A: 5 mM aqueous ammonium acetate solution; Mobile phase B: acetonitrile; Gradient: 10% B over 3 minutes, then 10% to 80% B over 14 minutes; Flow rate: 0.7 mL / minute. Temperature: room temperature; Detection: DAD at 215 nm; MS (+) ranges from 100-1500 daltons; Injection volume: 3 µL; Instrument: Waters 996 PDA.
[0704] [0704] Protocol M: Column: Phenomenex Luna, 150 x 3.0 mm, 5 µm; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in methanol (v / v); Gradient: 50% B for 1.5 minutes, 50% to 80% B for 8.5 minutes, then 80% B for 10 minutes; Flow rate: 0.75 mL / minute. Temperature: 45 ° C; Detection: DAD at 215 nm, 254 nm; MS (+) ranges from 90-2000 daltons; Injection volume: 10 µL; Instrument: Agilent 1200 LCMS.
[0705] [0705] Protocol N: Column: Phenomenex Luna C18 (2), 150 x 3.0 mm, 5 µm; Mobile phase A: 0.02% trifluoroacetic acid in water (v / v); Mobile phase B: 0.02% trifluoroacetic acid in acetonitrile (v / v); Gradient: 0% to 100% B over 23.5 minutes; Flow rate: 1.5 mL / minute. Temperature: uncontrolled; Detection: DAD at 210 nm; Injection volume: 10 mL; Instrument: Agilent 1100 HPLC.
[0706] [0706] Protocol O: Column: Column: Agilent Poroshell 300SB - C8, 75 x 2.1 mm, 2.6 µm; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient: 20% B to 45% B over 4 minutes; Flow rate: 1.0 mL / minute. Temperature: 60 ° C; Detection: 220 nm; MS (+) ranges from 400 - 2000 Da; Injection volume: 10 µL; Instrument: Agilent 1100 LC, Waters MicromassZQ MS. Decvolution was performed using MaxEnt1.
[0707] [0707] Protocol P: Column: Column: TSK - G3000SWxl gel, 300 x 7.8 mm, 10 µm; Mobile phase: phosphate buffer saline (PBS 1X), pH 7.4 with 2% acetonitrile; Isocratic; Flow rate: 1 mL / minute. Temperature: room temperature; Injection volume: 5 mL; Instrument: Agilent 1100 HPLC.
[0708] [0708] Protocol Q: Column: Waters Acquity UPLC HSS T3, C18, 2.1 x 50 mm, 1.7 µm; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient: 5% B over 0.1 minute, 5% to 95% B over 2.5 minutes, 95% B over 0.35 minute; Flow rate: 1.25 mL / minute. Temperature: 60 ° C; Detection: 200 - 450 nm; MS (+) ranges from 100-2000 daltons; Injection volume: 5 µL; Instrument: Waters Acquity.
[0709] [0709] Protocol Q1: Column: Waters Acquity UPLC HSS T3, C18, 2.1 x 50 mm, 1.7 m; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient: 5% B over 0.1 minute, 5% to 95% B over 1.5 minutes, 95% B over 0.35 minutes; Flow rate: 1.25 mL / minute. Temperature: 60 ° C; Detection: 200 - 450 nm; MS (+) ranges from 100-2000 daltons; Injection volume: 5 µL; Instrument: Waters Acquity.
[0710] [0710] Protocol Q2: Column: Xtimate C18, 2.1 x 30 mm, 3 µm; Mobile phase A: 0.1% trifluoroacetic acid in water (v / v); Mobile phase B: 0.1% trifluoroacetic acid in acetonitrile (v / v); Gradient: 10 to 80% B over 0.9 minutes, 80% B over 0.6 minutes; 100% B for 0.5 minutes ;: Flow rate: 1.2 mL / minute.
[0711] [0711] Protocol Q3: Column: Xtimate C18, 2.1 x 30 mm, 3 µm; Mobile phase A: 0.1% trifluoroacetic acid in water (v / v); Mobile phase B: 0.1% trifluoroacetic acid in acetonitrile (v / v); Gradient 0% to 60% B over 0.9 minutes, 60% B over 0.6 minutes; 100% B for 0.5 minutes; Flow rate: 1.2 mL / minute.
[0712] [0712] Protocol R: Column: Phenomenex Luna, 150 x 3.0 mm, 5 µm; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in methanol (v / v); Gradient: 5% B for 1.5 minutes, 5% to 100% B for 8.5 minutes, then 100% B for 1 minute; Flow rate: 0.75 mL / minute. Temperature: 45 ° C; Detection: DAD at 215 nm, 254 nm; MS (+) ranges from 150-2000 daltons; Injection volume: 10 µL; Instrument: 305 RP Agilent 1200 LCMS.
[0713] [0713] Protocol S: Column: Phenomenex Luna, 150 x 3.0 mm, 5 m; Mobile phase A: 0.1% trifluoroacetic acid in water (v / v); Mobile phase B: 0.1% trifluoroacetic in acetonitrile (v / v); Gradient: 5% B for 1.5 minutes, 5% to 95% B for 8.5 minutes, then 100% B for 1 minute; Flow rate: 1.0 mL / minute. Temperature: uncontrolled; Detection: DAD 210 nm; MS (+) ranges from 150-2000 daltons; Injection volume: 10 µL; Instrument: 305 RP Agilent 1100 HPLC.
[0714] [0714] Protocol T: Column: Atlantis dC18, 50 x 4.6 mm, 5 µm; Mobile phase A: 0.05% trifluoroacetic acid in water (v / v); Mobile phase B: 0.05% trifluoroacetic acid in acetonitrile (v / v); Gradient: 5% to 95% B over 4.0 minutes; then, it maintains 95% B for 1 minute; Flow rate: 2 mL / minute.
[0715] [0715] U Protocol: Column: Phenomenex Luna C18 (2), 150 x 3.0 mm, 5 µm; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient: 5% B for 1.5 minutes, 5% to 100% B for 8.5 minutes, then 100% B for 1 minute; Flow rate: 0.75 mL / minute. Temperature: 45 ° C; Detection: DAD at 215 nm, 254 nm; MS (+) ranges from 150-2000 daltons; Injection volume: 10 µL; Instrument: Agilent 1200 LCMS.
[0716] [0716] Protocol V: Column: HPLC - V Ultimate XB - C18, 50 x 3.0 mm, 3 µm; Mobile phase A: 0.225% trifluoroacetic acid in water (v / v); Mobile phase B: 0.225% trifluoroacetic acid in acetonitrile (v / v); Gradient: 30% to 90% B over 6 minutes; Flow rate: 1.2 mL / minute. Temperature: 40 ° C; Detection: DAD at 220 nm; Injection volume: 1 µL; Instrument: SHIMADZU.
[0717] [0717] Protocol W: Column: HPLC - V Ultimate XB - C18, 50 x 3.0 mm, 3 µM; Mobile phase A: 0.1% trifluoroacetic acid in water (v / v); Mobile phase B: 0.1% trifluoroacetic acid in acetonitrile (v / v); Gradient: 10% to 80% B over 6 minutes; Flow rate: 1.2 mL / minute. Temperature: 40 ° C; Detection: DAD at 220 nm; Injection volume: 3 µL; Instrument: SHIMADZU.
[0718] [0718] Protocol X: Column: YMC - Pack ODS-A, 150 x 4.6 mm, 5 µm; Mobile phase A: 0.1% trifluoroacetic acid in water (v / v); Mobile phase B: 0.1% trifluoroacetic acid in acetonitrile (v / v); Gradient: 10% to 80% B over 6 minutes; Flow rate: 1.2 mL / minute. Detection: DAD at 220 nm. Temperature: 40 ° C; Injection volume: 3 µL; Instrument: SHIMADZU.
[0719] [0719] Protocol Y: Column: YMC - Pack ODS-A, 150 x 4.6 mm, 5 µm; Mobile phase A: 0.1% trifluoroacetic acid in water (v / v); Mobile Phase B: 0.1% trifluoroacetic acid in acetonitrile (v / v); Gradient: 0% to 95% B over 10 minutes, then 95% B over 5 minutes; Flow rate: 1.5 mL / minute; Detection: DAD at 220 nm; Instrument: Agilent 1100.
[0720] [0720] Protocol Z: Column: Xtimate C18, 2.1 x 30 µm, 3 mM; Mobile phase A: 0.1% trifluoroacetic acid in water (v / v); Mobile phase B: 0.1% trifluoroacetic acid in acetonitrile (v / v); Gradient: 0% to 60% B over 2 minutes; Flow rate: 1.2 ml / min. Temperature: 50 ° C; Detection: 220 nm, MS (+) ranges from 100-1000 daltons; Injection volume: 1 µL; Instrument: SHIMADZU.
[0721] [0721] AB Protocol: Column: Phenomenex Luna C18 (2), 150 x 2.0 mm, 5 µm; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient: 5% to 100% B over 10 minutes, then 100% B over 2 minutes; Flow rate: 0.5 mL / minute. Temperature: 25 ° C; Detection: DAD at 210 nm, 254 nm; MS (+) ranges from 150-2000 daltons; Injection volume: 5 µL; Instrument: Agilent 1100 LCMS.
[0722] [0722] BB Protocol: Column: Phenomenex Luna C18 (2), 150 x 2.0 mm, 5 µm; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient: 5% B for 2.0 minutes, 5% to 100% B for 12 minutes and 100% B for 2 minutes, then 100% to 5% B for 1.5 min; Flow rate: 0.75 mL / minute. Temperature: 25 ° C; Detection: DAD at 215 nm, 254 nm; MS (+) ranges from 150-2000 daltons; Injection volume: 5 µL; Instrument: Agilent.
[0723] [0723] CB Protocol: Column: Waters XBridge C18, 4.6 x 50 mm, 5 µm; Mobile phase A: 0.03% ammonium hydroxide in water (v / v); Mobile phase B: 0.03% ammonium hydroxide in acetonitrile (v / v); Gradient: 5% to 95% B for 4.0 minutes, then 95% B for 1 minute; Flow rate: 2 mL / minute. Temperature: 25 ° C; Detection: DAD at 215 nm, MS (+) ranges from 160-1000 daltons; Injection volume: 4 µL; Instrument: Waters ZQ / Alliance 2795 HPLC.
[0724] [0724] DB Protocol: Column: Waters Atlantis dC18, 4.6 x 50 mm, 5 µm; Mobile phase A: 0.05% trifluoroacetic acid in water (v / v); Mobile phase B: 0.05% trifluoroacetic acid in acetonitrile (v / v); Gradient: 5.0% a / 95% B for 4.0 minutes, then 95% B for 1 minute. Flow rate: 2 mL / minute.
[0725] [0725] EB Protocol: Column: XBridge RP18, 2.1 x 50 mm, 5 µm; Mobile phase A: 0.02% ammonium hydroxide in water (v / v); Mobile phase B: 0.02% ammonium hydroxide in acetonitrile (v / v); Gradient of 10% to 80% B over 6 minutes, then 80% over 2 minutes; Flow rate: 1.2 mL / minute. Detection: DAD at 220 nm; Temperature: 50 ° C.
[0726] [0726] FB Protocol: Column: Phenomenex Luna C18 (2), 150 x 2.0 mm, 5 µm; Mobile phase A: 0.1% formic acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient: 5% B over 2.0 minutes, 5% to 100% B over 10 minutes, and 100% B over 2 minutes; Flow rate: 0.50 mL / minute. Temperature: 25 ° C; Detection: DAD at 215 nm, 254 nm; MS (+) ranges from 150-2000 daltons; Injection volume: 5 µL; Instrument: Agilent 1200 LCMS.
[0727] [0727] In some cases, some minor changes for LC - MS analyzes and HPLC conditions have been made such as, but not limited to, the change in gradient or flow rate, which is indicated by the * symbol.
[0728] [0728] HPLC conditions used for Purification
[0729] [0729] Method A: Column: Phenomenex Lux Amylose - 2, 250 x 21.2 mm, 5 µm; Mobile phase A: heptane; Mobile phase B: ethanol (denatured); Gradient: 5% to 100% B over 6 min; Flow rate: 27 mL / minute; Detection: DAD at 210-360 nm; MS (+) ranges from 150-2000 daltons; Instrument: Waters FractionLynx.
[0730] [0730] Method B: Column: Phenomenex Luna C18 (2), 150 x 21.2 mm, 5 µm; Mobile phase A: 0.02% acetic acid in water; Mobile phase B: 0.02% acetic acid in acetonitrile; Gradient: 5% B over 1.5 minutes, 5% to 45% B over 8.5 minutes; Flow rate: 27 mL / minute; Detection: DAD at 215 nm, 254 nm; MS (+) ranges from 150-2000 daltons; Instrument: Waters FractionLynx.
[0731] [0731] Method C: Column: Phenomenex Luna C18, 100 x 30 mm, 10 µM; Mobile phase A: 0.02% trifluoroacetic acid in water (v / v); Mobile phase B: 0.02% trifluoroacetic acid in methanol (v / v); Gradient: 10% to 90% B over 20 minutes; Flow rate: 20 mL / minute. Temperature: uncontrolled; Detection: DAD at 210 nm, 254 nm; Injection volume: variable; Instrument: Gilson.
[0732] [0732] Method D: Column: Phenomenex Synergi Max-RP, 150 x 21.2 mm, 4 µm; Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile; Gradient: 30% B for 1.5 minutes, 30% to 60% B for 8.5 minutes, 60 to 100% B for 0.5 minutes, then 100% B for 2 minutes; Flow rate: 27 mL / minute; Detection: DAD at 210-360 nm; MS (+) ranges from 150-2000 daltons; Instrument: Waters FractionLynx.
[0733] [0733] Method E1: Column: Phenomenex Luna C18 (2), 150 x 21.2 mm, 5 µm; Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile; Gradient: 40% B for 1.5 minutes, 40% to 80% B for 8.5 minutes, 80 to 100% B for 0.5 minutes, then 100% B for 2 minutes; Flow rate: 27 mL / minute; Detection: Detection: DAD at 210-360 nm; MS (+) ranges from 150-2000 daltons; Instrument: Waters FractionLynx LCMS.
[0734] [0734] Method E2: Column: Phenomenex Luna Phenyl-hexyl, 150 x 21.2 mm, 5 µm. The rest of the protocols are identical to those described for Method E1.
[0735] [0735] Method F: Column: Phenomenex Synergi Max-RP, 150 x 21.2 mm, 4 µm; Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in methanol; Gradient: 44% B over 1.5 minutes, 44% to 77% B over 8.5 minutes, then 77% B over 10 minutes; Flow rate: 27 mL / minute; Detection: DAD at 210-360 nm; MS (+) ranges from 150-2000 daltons; Instrument: Waters FractionLynx LCMS.
[0736] [0736] Method G: Column: PrincetonSFC 2 - ethylpyridine, 250 x 21.2 mm, 5 µm; Mobile phase A: heptane; Mobile phase B: ethanol (denatured); Gradient: 1% B over
[0737] [0737] Method H: Column: Phenomenex Luna C18 (2), 150 x 21.2 mm, 5 µm; Mobile phase A: 0.02% acetic acid in water; Mobile phase B: 0.02% acetic acid in acetonitrile; Gradient: 20% B over 1.5 minutes, 20% to 60% B over 10.5 minutes; Flow rate: 27 mL / minute; Detection: DAD at 210-360 nm; MS (+) ranges from 150-2000 daltons; Instrument: Waters FractionLynx LCMS.
[0738] [0738] Method I: Column: Phenomenex Luna C18 (2), 150 x 21.2 mm, 5 µm; Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in methanol; Gradient: 40% B over 1.5 minutes, 40% to 70% B over 8.5 minutes, then 70% B over 10 minutes; Flow rate: 27 mL / minute; Detection: DAD at 210-360 nm; MS (+) ranges from 150-2000 daltons; Instrument: Waters FractionLynx LCMS.
[0739] [0739] Method J: Column: Phenomenex Luna C18, 100 x 30 mm, 5 µm; Mobile phase A: 0.02% trifluoroacetic acid in water (v / v); Mobile phase B: 0.02% trifluoroacetic acid in acetonitrile (v / v); Gradient: 10% to 90% B over 20 minutes; Flow rate: 20 mL / minute. Temperature: uncontrolled; Detection: DAD at 210 nm, 254 nm; Injection volume: variable; Instrument: Gilson.
[0740] [0740] Method K: Column: Phenomenex Luna C18 (2), 150 x 21.2 mm, 5 µm; Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile; Gradient: 20% B for 1.5 minutes, 20% to 50% B for 8.5 minutes, 50 to 100% B for 0.5 minutes, then 100% B for 2 minutes; Flow rate: 27 mL / minute; Detection: Detection: DAD at 210-360 nm; MS (+) ranges from 150-2000 daltons; Instrument: Waters Fraction Lynx LCMS.
[0741] [0741] Method L: Column: Phenomenex Luna C18 (2), 150 x 21.2 mm, 5 µm; Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile; Gradient: 30% B for 1.5 minutes, 30% to 50% B for 8.5 minutes, 50 to 100% B for 0.5 minutes, then 100% B for 2 minutes; Flow rate: 27 mL / minute; Detection: Detection: DAD at 210-360 nm; MS (+) ranges from 150-2000 daltons; Instrument: Waters Fraction Lynx LCMS.
[0742] [0742] Method M: Column: Waters Sunfire C18, 19 x 100 mm, 5 µm; Mobile phase A: 0.05% trifluoroacetic acid in water (v / v); Mobile phase B: 0.05% trifluoroacetic acid in acetonitrile (v / v); Gradient: 0 to 100% over 8.5 minutes. Flow rate: 25 mL / minute. Detection: DAD at 215 nm. MS (+) ranges from 160-1000 daltons; Instrument: Waters FractionLynx.
[0743] [0743] Method N: Column: Waters Sunfire C18, 19 x 100 mm, 5 µm; Mobile phase A: 0.05% formic acid in water (v / v); Mobile phase B: 0.05% formic acid in acetonitrile (v / v); Gradient: 0 to 100% over 8.5 minutes. Flow rate 25 mL / minute. Detection: DAD at 215 nm. MS (+) ranges from 160-1000 daltons; Instrument: Waters FractionLynx.
[0744] [0744] Method O: Column: Phenomenex Luna C18, 21.2 x 150 mm, 5 µm; Mobile phase A: 0.1% formic acid in water (v / v) acid in water (v / v); Mobile phase B: 0.1% formic acid in acetonitrile (v / v); Gradient (v / v); Gradient 20% B over 1.5 minutes, 20% to 40% B over 8.5 minutes, 40 to 100% B over 0.5 minutes, then maintained at 100% B for 1.5 minutes . Flow rate: 27 mL / minute. Detection: DAD at 210-360 nm; MS (+) ranges from 150-2000 daltons; Instrument: Waters FractionLynx.
[0745] [0745] Method P: Column: Phenomenex GeminiC18, 21.2 × 250 mm, 5 µm; Mobile phase A: 0.225% ammonium hydroxide in water (pH 10) (v / v); Mobile phase B: 0.225% ammonium hydroxide in acetonitrile (v / v); Gradient: 45% to 85% B over 10 minutes. Flow rate 35 mL / minute. Detection: DAD at 220 nm. MS (+) ranges from 100-1200 daltons; Instrument: Shimadzu MS Trigger.
[0746] [0746] Method Q: Column: Column: Phenomenex Synergi C18, 50 x 250 mm, 10 µm; Mobile phase A: 0.1% trifluoroacetic acid in water (v / v). Mobile phase B: Acetonitrile; Gradient of 10% to 40% B over 25 minutes. Flow rate 100 mL / minute. Detection: UV / Vis 220 nm; Instrument: Shimadzu LC - 8A.
[0747] [0747] Method R: Column: Phenomenex Luna C18 (2), 250 x 21.2 mm, 5 µm; Mobile phase A: 0.1% TFA in water (v / v); Mobile phase B: 0.1% TFA in acetonitrile (v / v); Gradient: 10% to 100% over 30 minutes; Variable flow rate.
[0748] [0748] In some cases, some minor changes to purification conditions have been made such as, but not limited to, a change in the gradient or flow rate, which is indicated by the * symbol.
[0749] [0749] General Procedures
[0750] [0750] General Procedure A: removal of Fmoc with diethylamine or piperidine. To a solution of the compound containing Fmoc in dichloromethane or N, N-dimethylformamide (also referred to as DMF), an equal volume of diethylamine or piperidine was added. The progress of the reaction was monitored by LC-MS (or HPLC or TLC). The solvents were removed in vacuo, and in some cases, the residue was azeotroped one to four times with heptane. The residue was generally diluted with dichloromethane and a small amount of methanol, before being reduced down on silica and purified by chromatography on silica gel, eluting with methanol in dichloromethane (or another suitable mixture of solvents) to provide the desired material (or material crude was used as such).
[0751] [0751] General Procedure B: Boc removal or t-Bu ester cleavage using trifluoroacetic acid. To a solution of the compound containing Boc or compound containing tert-butyl ester in dichloromethane at 0 ° C (or at room temperature), trifluoroacetic acid was added to provide a 1: 4 ratio of trifluoroacetic acid: dichloromethane. The progress of the reaction was monitored by LC-MS (or HPLC or TLC). The solvents were removed in vacuo. The residue was azeotroped three times with heptane to provide the desired material.
[0752] [0752] General Procedure C: Boc removal or tert-butyl ester cleavage (also refers to t-Bu ester) using hydrochloric acid in dioxane. For any solution of compound containing Boc or compound containing tert-butyl ester in dioxane (or in some cases no solution, or other relevant solvent) a 4 M solution of hydrochloric acid in dioxane was added. The progress of the reaction was monitored by LC-MS (or HPLC or TLC). The reaction was concentrated in vacuo and, in some cases, azeotroped one to four times with heptanes.
[0753] [0753] General Procedure D: coupling with O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium (HATU). To a stirred solution of amine (1.0 eq.) And acid (1.0-2.0 eq.) In dichloromethane, N, N-dimethylformamide (also referred to as DMF), or a mixture of both, HATU (1 , 0-2.0 eq.) Was added followed by triethylamine (2.0-4.0 eq.) Or diisopropylethylamine (2.0-4.0 eq., Also referred to as Hunig's base). The progress of the reaction was monitored by LC-MS (or HPLC or TLC); the reaction was usually completed within three hours. The solvents were removed in vacuo. The residue was purified by reverse phase chromatography or silica gel or, in some cases, azeotroped three times with heptanes, diluted with a small amount of ethyl acetate before being reduced down to silica or C18 bound silica and purified by chromatography on reverse phase or silica gel.
[0754] [0754] General Procedure E: coupling with N- [6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] -L-valyl-N5-carbamoyl-N- [4 - ({[(4-nitrophenoxy) carbonyl] oxy} methyl) phenyl] - L-ornithinamide (MalcValCitPABC-PNP). To a mixture of loading amine (1 eq.) And N- [6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] -L-valyl-N5-carbamoyl- N- [4 - ({[(4-nitrophenoxy) carbonyl] oxy} methyl) phenyl] -L-ornithinamide (MalcValCitPABC-PNP, Pat. Eur. (1994), EP624377, 1.0-2.0 eq .) in N, N-dimethylformamide or dimethylacetamide (also referred to as DMA), pyridine (0.0-4.0 eq.), diisopropylethylamine (0.0-4.0 eq.), 2,6-dimethylpyridine (0 , 0-4.0 eq., Also referred to as 2,6-Luditine) and 1-hydroxybenzotriazole hydrate (0.01-1.1 eq. Also referred to as HOBT) or 3H- [1,2,3] triazole [ 4,5-b] pyridin-3-ol (0.01-1.1 eq., Also referred to as HOAT) was added. After stirring at 40 ° C-50 ° C for 1-48 hours, the reaction mixture was concentrated in vacuo and azeotroped three times with heptane.
[0755] [0755] General procedure F: conjugation of commercial HERCEPTIN® antibody with ligand-load by means of internal disulfites. Commercially available HERCEPTIN® antibody (Genentech, Inc), was dialyzed in Dulbecco's Phosphate Buffered Saline (DPBS, Lonza). The dialysed antibody was reduced with the addition of x equivalents of tris (2-carboxyethyl) phosphine hydrochloride (TCEP, 5 mM in distilled water) and diluted to 15 mg / mL of final antibody concentration using DPBS, 5 mM of 2, 2 ', 2' ', 2' '' - (ethane-1,2-diyldinitrile) tetraacetic acid (EDTA), pH 7.0-7.4 (buffer A). The reaction was incubated at 37 ° C for 1-2 hours and then cooled to room temperature. The conjugation was performed by adding y equivalents of binder-filler (5-10 mM in dimethylacetamide (DMA)). DMA was added to achieve 10-20% (v / v) of the total organic solvent component in the final reaction mixture, and buffer A added to achieve 10 mg / ml of final antibody concentration. The reaction was incubated for 1-2 hours at room temperature. The reaction mixture was then changed from buffer to DPBS (pH 7.4), using GE Healthcare Sephadex G-25 M buffer exchange columns according to the manufacturer's instructions. The crude material was purified by size exclusion chromatography (SEC) using a GE AKTA Explorer system with a Superdex GE column and PBS (pH 7.4) as eluent.
[0756] [0756] General Procedure G: Conjugation reactions were performed on the top of a centrifugal ultrafiltration device, such as Amicon Ultra 50k Ultracel filters (part # UFC805096, GE). A 132 mM L-cysteine stock solution was prepared in PBS containing 50 mM EDTA. This solution (50 µL) was added to a mixture of the respective mutant antibody (5 mg) in 950 µL PBS containing 50 mM EDTA. The final cysteine concentration in the reaction mixture was 6.6 mM. After allowing the reaction to rest at room temperature (about 23 ° C) for 1.5 hours, the reaction tube was centrifuged to concentrate the material at approximately 100 µL. The mixture was diluted to 1 ml with PBS containing 50 mM EDTA. This process was repeated 4 times in order to remove the entire cysteine reducer. The resulting material was diluted to 1 ml in PBS containing 50 mM EDTA and treated with 16 µL of a 5 mM solution of maleimide binder-filler (from Table 18A in dimethyl acetamide (DMA) (about 5 equivalents) After resting at room temperature (about 23 ° C) for 1.5 hours, the reaction tube was centrifuged to concentrate the material to approximately 100 µL. The mixture was diluted to 1 ml with PBS. This process was repeated 2 times to remove excess maleimide reagent. Antibody conjugates were generally purified by size exclusion chromatography (SEC) using a GE AKTA Explorer system with a GE Superdex200 column and PBS (pH 7.4) as Drug loading to the desired conjugation site was determined using a variety of methods, including mass spectrometry (MS), reverse phase HPLC, and hydrophobic interaction chromatography (HIC), as described elsewhere The ditch r reported (in Tables 19A and 19B) is generally obtained by LC-MS under conditions of reduction.
[0757] [0757] General Procedure H: A 20 mM solution of TCEP (usually 50 to 100 molar equivalents) was added to the antibody (typically 5 mg) such that the final antibody concentration was 5 mg / ml in PBS containing 50 mM EDTA. After allowing the reaction to rest at 37 ° C for 1.5 hours, the antibody was buffered in PBS containing 50 mM EDTA using a 50 kD MW rotary cut-off concentration device (3 x 3 mL wash, 10x concentration per cycle). Alternative methods, such as TFF or dialysis are also useful in particular circumstances. The resulting antibody was resuspended in 1 ml of PBS containing 50 mM EDTA and treated with a freshly prepared 50 mM DHA solution (dehydroascorbate) in 1: 1 PBS / EtOH (final DHA concentration is typically 1 mM ) and allowed to rest at 4 ° C overnight. The antibody / DHA mixture was buffered in PBS containing 50 mM EDTA, using a 50 kD MW spin cut-off concentration device (3 x 3 mL wash, 10x concentration per cycle). The resulting antibody was resuspended in 1 ml of PBS containing 50 mM EDTA and treated with 10 mM maleimide ligand-loading in DMA (typically 5-10 equivalents). After resting for 1.5 hours, the material was changed from buffer (as above) in 1 ml of PBS (3 x 3 ml washes, 10x concentration per cycle). SEC purification (as described above) was performed as needed to remove any aggregate material.
[0758] [0758] General Procedure I: The initial ligand-load conjugation was performed using the method previously described (General Procedure F). The resulting antibody-drug-conjugate was changed from buffer to a 50 mM borate buffer (pH 9.2) using an ultrafiltration device (50 kd MW cut). The resulting solution was heated at 37 ° C for 24 hours (for maleimide-Peg linkers) or at 45 ° C for 48 hours (for maleimide-caproil linkers).
[0759] [0759] General Procedure J: The pentafluorophenyl esters were conjugated to the antibody shown following the procedure previously described in WO2012007896 A1.
[0760] [0760] General Procedure K: The conjugation of amino-alkyl ligands was carried out through enzyme-mediated binding, as described in WO2012059882 A2.
[0761] [0761] General Procedure L: N - [(9H-fluoren-9-ylmethoxy) carbonyl] -2-methylalanyl-N- [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -2-carboxy-1-methoxypropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamine (prepared in the same manner as # 136 ) was coupled to the relevant amino acid or amine moiety using HATU (1.0-2.0 eq.) in the presence of Hunig's base (1.0-5.0 eq.) in a solution of DMF, dichloromethane, or in some cases, a solution of both (or a solution of one or more solvents). The reaction was monitored by LC-MS (or TLC or HPLC). The reaction was concentrated in vacuo and generally purified by silica chromatography or by preparative HPLC. The Fmoc protection was then removed as described in general procedure A followed by concentration in vacuo and purified by chromatography on silica or by preparative HPLC.
[0762] [0762] General Procedure M: # 151 was coupled to the relevant amine using HATU (1.0-2.0 eq., Or another suitable coupling reagent) in the presence of Hunig's base (1.0-5.0 eq. ) in a solution of DMF, dichloromethane, or in some cases, a solution of both (or a solution of one or more solvents). The reaction was monitored by LC-MS (or TLC or HPLC). The reaction was concentrated in vacuo.
[0763] [0763] General Procedure N. Acid 1- (9H-fluorene-9-yl) -3-oxo-2,7,10,13,16,19,22- heptaoxa-4-azapentacosan-25-oic (or other Fmoc-AmPegXC2-COOH) was coupled to the relevant cytotoxic pentapeptide (or the cytotoxic pentapeptide containing a protecting group in a reactive portion other than N-terminal), using HATU (1.0-2.0 eq., Or other reagent coupling), in the presence of Hunig's base (1.0-5.0 eq. or other suitable base) in a solution of DMF, dichloromethane, or in some cases, a solution of both (or a solution of one or more solvents). The reaction was monitored by LC-MS (or TLC or HPLC). The reaction was concentrated in vacuo. Deprotection of Fmoc was performed according to general procedure A. In some cases, a second deprotection was performed in order to remove a protecting group in a reactive portion on the cytotoxic pentapeptide, using a general procedure B (or other relevant procedure known in based on the protection group). The reaction was concentrated in vacuo and purified by chromatography on silica or by preparative HPLC.
[0764] [0764] General Procedure O. The appropriate Fmoc-AmPegXC2-COOH is linked to the relevant cytotoxic pentapeptide (or the cytotoxic pentapeptide containing a protecting group in a reactive portion other than N-terminal) and deprotection of Fmoc is performed according to the general procedure N. The reaction is concentrated in vacuo and then purified by chromatography on silica or preparative HPLC (or the crude material can be used as such). The appropriate PABC sequence (such as mcValCitPABC, or derived from) is then installed according to general procedure E. In some cases, if a protecting group is present in the cytotoxic pentapeptide portion of the molecule's deprotection is , then performed using general procedure A or general procedure B (or other relevant procedure known in the literature based on the protection group). The reaction is concentrated in vacuo and purified by chromatography on silica or by preparative HPLC.
[0765] [0765] General Procedure P. Procedure E is followed by replacing mcValCitPABC-PNP with MalPeg3C2ValCitPABC-PNP (prepared in a similar manner to mcValCitPABC-PNP).
[0766] [0766] General Procedure Q: The appropriate Fmoc-AmPegXC2-COOH is linked to the relevant cytotoxic pentapeptide (or cytotoxic pentapeptide containing a protection group in a reactive portion other than N-terminal) and Fmoc deprotection is performed as described in the procedure general N. The reaction is concentrated in vacuo and then purified by silica chromatography or by preparative HPLC (or the crude material can be used as such). To a stirring solution of this residue in DMF at 0 ° C (or at a slightly higher temperature, in some cases), bromoacetic acid (1.0-2.0 eq.) Was added followed by Hunig's base (1, 0-5.0 eq.) And HATU (1.0-2.0 eq.). The reaction was allowed to warm up to room temperature and stir at room temperature while being monitored by LC-MS (or TLC or HPLC). The reaction was concentrated in vacuo and purified by preparative HPLC.
[0767] [0767] General Procedure R. Procedure E followed by replacing mcValCitPABC-PNP, with N- (6 - {[(9H-fluoren-9-ylmethoxy) carbonyl] amino} hexanoyl) -L-valyl-N ~ 5 ~ -carbamoyl -N- [4 - ({[(4-nitrophenoxy) carbonyl] oxy} methyl) phenyl] -L-ornithinamide (prepared in a manner similar to mcValCitPABC-PNP). Deprotection of Fmoc was then performed (general procedure B) followed by preparative HPLC purification.
[0768] [0768] General Procedure S. To a stirring solution of 6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -hexanal (1.0-3.0 eq.) In methanol, the relevant cytotoxic pentapeptide (1.0 eq.) was added followed by formic acid. The reaction was allowed to stir at room temperature for 1-40 minutes, followed by the addition of sodium
[0769] [0769] General Procedure T. 4- [3-oxo-3- (2-oxoazetidin-1-yl) propyl] anilinium is prepared as described in the literature (Bioorganic and Medicinal Chemistry Letters, 2012, vol 22, # 13, 4249 -4253), which is then coupled to bis (pentafluorophenyl) 3,3 '- [ethane-1,2-diylbis (oxy)] dipropanoate using HATU in dichloromethane, followed by coupling with the desired cytotoxic pentapeptide.
[0770] [0770] General Procedure U. Fmoc-ValCitPABC-PNP is coupled to the desired cytotoxic pentapeptide following general procedure E, and then Fmoc is removed following general procedure A. This residue is then coupled to the acid [2 -oxo-2 - ({4- [3-oxo-3- (2-oxoazetidin-1-yl) propyl] phenyl} amino) ethoxy] acetic (which is prepared by coupling 4- [3-oxo-3- (2-oxoazetidin-1-yl) propyl] anilinium with 1,4-dioxane-2,6-dione following general procedure D). The material is then purified by preparative HPLC.
[0771] [0771] General Procedure V. Bis (pentafluorophenyl) 3,3 '- [ethane-1,2-diylbis (oxy)] dipropanoate or bis (pentafluorophenyl) -4,7,10,13,16-pentaoxanonadecane- 1,19 -diate is coupled to the desired cytotoxic pentapetide (or in some cases coupled to the desired cytotoxic pentapeptide containing a protection group in a reactive portion other than the N-terminal) following general procedure D. If a protection group is present, the group protection is then removed (using relevant procedures described in the literature). The material is then purified by preparative HPLC.
[0772] [0772] General Procedure W. 4 - ({[(4-nitrophenoxy) carbonyl] oxy} methyl) phenyl 1- (9H-fluorene-9-yl) -3-oxo-2,7,10-trioxa-4- azatridecan-13-oate is coupled to the desired cytotoxic pentapeptide following the general procedure E. Fmoc is removed following the general procedure A. Bis (pentafluorophenyl) 3,3 '- [ethane-1,2-diylbis (oxy)] dipropanoate is coupled to this residue following general procedure D. The material is then purified by preparative HPLC.
[0773] [0773] General Procedure X1. N - [(9H-fluoren-9-ylmethoxy) carbonyl] -L-alanyl-L-alanyl- N ~ 1 ~ - [4 - ({[(4-nitrophenoxy) carbonyl] oxy} methyl) phenyl] -N ~ 4 ~ -trityl-L-aspartamide is coupled to the desired cytotoxic pentapeptide following general procedure E. Fmoc is removed following general procedure A and the trityl protecting group is removed following general procedure B. Bis (pentafluorophenyl) 3.3 ' - [ethane-1,2-diylbis (oxy)] dipropanoate is coupled to this residue following general procedure D.
[0774] [0774] General Procedure X2. N- {3- [2- (3-ethoxy-3-oxopropoxy) ethoxy] propanoyl} -L-valyl-N ~ 5 ~ -carbamoyl-N- [4 - ({[(4-nitrophenoxy) carbonyl] oxy} methyl) phenyl] -L-ornithinamide is coupled to the desired cytotoxic pentapeptide following general procedure E.
[0775] [0775] General Procedure X3. N - [(9H-fluoren-9-ylmethoxy) carbonyl] -D-valyl-N ~ 5 ~ - carbamoyl-N- [4 - ({[(2-carboxypropan-2-yl) carbamoyl] oxy} methyl) phenyl ] -L-ornithinamide is coupled to # 50 following general procedure D in DMSO and acetonitrile. Fmoc is removed following general procedure A, followed by coupling with bis (pentafluorophenyl) 3,3 '- [ethane-1,2-diylbis (oxy)] dipropanoate using Hunig's base in acetonitrile. The material is purified by preparative HPLC.
[0776] [0776] General Procedure X4. N- [1- (9H-fluoren-9-yl) -3,5,12-trioxo-2,7,10-trioxa-4-azadodecan-12-yl] -2-methylalanine is coupled to # 250 following the general procedure D in acetonitrile. Fmoc is removed following general procedure A, followed by coupling with bis (pentafluorophenyl) 3,3 '- [ethane-1,2-diylbis (oxy)] dipropanoate using Hunig's base in acetonitrile. The material is purified by preparative HPLC.
[0777] [0777] General Procedure X5. L-valyl-N ~~ 5-carbamoyl-N- [4- (hydroxymethyl) phenyl] - L-ornithinamide is coupled to N ~ 2 ~ -acetyl-N ~ 6 ~ - (tert-butoxycarbonyl) -L-lysine following the general procedure D. This resulting residue is coupled with Hunig-based bis (4-nitrophenyl) carbonate in DMF, followed by coupling with the desired cytotoxic pentapeptide following general procedure E. Boc deprotection is then carried out according to with general procedure B in acetonitrile.
[0778] [0778] In some cases, minor changes to the reaction conditions have been made such as, but not limited to the order of reagent and addition of reagents and or the amount of reagent or reagent that is indicated by the symbol *. In addition, these general procedures are provided as examples only and are non-limiting.
[0779] [0779] In addition to the General Procedures provided above, the relevant dolastatin and auristatin references include the following: Petit et al. J. Am. Chem. Soc.
[0780] [0780] MS Analysis and Sample Preparation
[0781] [0781] The samples were prepared for LC-MS analysis, combining about 20 µL of sample (about 1 mg / mL ADC in PBS) with 20 µL of 20 mM dithiothreitol (DTT). After letting the mixture rest at room temperature for 5 minutes, the samples were analyzed according to the O protocol.
[0782] [0782] The following calculation was performed in order to establish the total charge (DAR) of the conjugate:
[0783] [0783] Loading = 2 * [LC1 / (LC1 + LC0)] + 2 * [HC1 / (HC0 + HC1 + HC2 + HC3)] +
[0784] [0784] 4 * [HC2 / (HC0 + HC1 + HC2 + HC3)] + 6 * [HC3 / (HC0 + HC1 + HC2 + HC3)]
[0785] [0785] where the variables indicated are the relative abundance of: LC0 = unloaded light chain, LC1 = single loaded light chain, HC0 = unloaded heavy chain, HC1 = single loaded heavy chain, HC2 = double loaded heavy chain, and HC3 = chain heavy loaded triple.
[0786] [0786] LC-MS conditions used are Protocol F for the retention time less than one minute and Protocol H for the remaining experiments, unless otherwise indicated.
[0787] [0787] Preparation of N - [(9H-Fluoren-9-ylmethoxy) carbonyl] -N-methyl-L-valyl-N- [(2R, 3S, 4S) -1-carboxy-2-methoxy-4-methylhexan -3-yl] -N-methyl-L-valinamide (# 8)! "#
[0788] [0788] Step 1. Synthesis of benzyl [(2S, 3S) -1-hydroxy-3-methylpentan-2-yl] methylcarbamate (# 1). To a solution of N - [(benzyloxy) carbonyl] -N-methyl-L-isoleucine (52.37 g, 187.5 mmol, 1 eq.) In tetrahydrofuran (524 mL, 0.35 M) was added with borane complex -tetrahydrofuran (1 M in tetrahydrofuran, 375 ml, 375 mmol, 2 eq.) slowly over 1 hour and the reaction was allowed to stir for 18 hours at room temperature. The reaction was cooled to 0 ° C and water (30 ml) was added over 30 minutes. The reaction mixture was diluted with 1 M aqueous sodium carbonate solution (100 ml) and tert-butyl methyl ether (250 ml). The aqueous layer was re-extracted with tert-butyl methyl ether (100 ml). The combined organic layers were washed with 1 M aqueous sodium carbonate solution (100 ml), washed with brine (200 ml), dried over magnesium sulfate, filtered, and concentrated in vacuo to provide # 1 (48.44 g , 97% yield) as a pale yellow oil, which was used in the next step without further purification. 1H NMR (400 MHz, DMSO-d6), it is assumed to be a mixture of rotamers: δ 7.26-7.41 (m, 5H), [5.06 (AB quartet, JAB = 12.9 Hz, vAB = 22.8 Hz) and 5.06 (AB quartet, JAB = 9.0 Hz, vAB = 9.0 Hz), total 2H], [4.65 (t, J = 5.3 Hz) and 4, 59 (t, J = 5.4 Hz), 1H total], 3.67-3.80 (m, 1H), 3.51-3.60 (m, 1H), 3.41-3.51 ( m, 1H), 2.75 and 2.71 (2 s, 3H total), 1.49-1.64 (br m, 1H), 1.24-1.37 (br m, 1H), 0, 90-1.02 (br m, 1H), 0.74-0.87 (m, 6H).
[0789] [0789] Step 2. Synthesis of benzyl methyl [(2S, 3S) -3-methyl-1-oxopentan-2-yl] carbamate (# 2). To a solution of # 1 (8.27 g, 31.2 mmol, 1 eq.) In dimethyl sulfoxide (41.35 mL, 0.75 M), was added triethylamine (8.70 mL, 64.0 mmol , 2.05 eq.) And the mixture was cooled to 0 ° C. Sulfur trioxide pyridine complex (10.18 g, 63.96 mmol, 2.05 eq.) Was then added in small portions, keeping the internal temperature below 8 ° C. The reaction was allowed to reach room temperature and was stirred for 18 hours. The reaction was poured into water (100 ml) and tert-butyl methyl ether (100 ml). The aqueous layer was re-extracted with tert-butyl methyl ether (50 ml) and the combined organic layers were washed with brine (100 ml), dried over magnesium sulfate, filtered, concentrated in vacuo and purified by chromatography on silica gel ( Gradient: 10% to 60% ethyl acetate in heptane) to provide # 2 (7.14 g, 87%) as a colorless oil. 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ 9.61 (s, 1H), 7.26-7.42 (m, 5H), 5.01- 5.13 (m, 2H), 4.04 - 4.12 (m, 1H), 2.86 and 2.82 (2 s, 3H total), 1.94-2.11 (br m, 1H) , 1.26-1.42 (br m, 1H).
[0790] [0790] Step 3. Synthesis of tert-butyl (3R, 4S, 5S) -4 - {[(benzyloxy) carbonyl] (methyl) amino} -3-hydroxy-5-methylheptanoate (# 3). Diisopropylamine lithium was prepared by adding n-butyllithium (2.5 M solution in tetrahydrofuran, 35.9 mL, 89.8 mmol, 1.4 eq.) To a diisopropylamine solution (13.8 mL, 96, 3 mmol, 1.5 eq.) In tetrahydrofuran (50 ml, 1.3 M) at -78 ° C. After 1 hour, tert-butyl acetate (15.7 mL, 116 mmol, 1.8 eq.) Was added dropwise and the reaction mixture was stirred for an additional 1.5 hours, while allowed to warm slowly to -20 °. Ç. The reaction mixture was cooled again to -78 ° C and a solution of aldehyde # 2 (16.9 g, 64.2 mmol, 1 eq.) In tetrahydrofuran (10 ml) was added. The reaction mixture was stirred for 1.5 hours and then quenched by the addition of water (100 ml). After extraction with diethyl ether (2 x 100 mL), the combined organic layers were dried over sodium sulfate, filtered, concentrated in vacuo and purified by chromatography on silica gel (Gradient: 0% to 20% acetone in heptane) to provide # 3 (8.4 g, 34%) as a colorless oil. LC-MS: m / z 402.4 [M + Na +], retention time = 3.91 minutes; 1H NMR (400 MHz, DMSO-d6), it is assumed to be a mixture of rotamers: δ 7.27-7.39 (m, 5H), 5.01-5.12 (m, 2H), [4, 93 (d, J = 7.2 Hz) and 4.98 (br d, J = 7.2 Hz), 1H total], 4.03-4.15 (br m, 1H), 3.68-3 , 85 (br m, 1H), 2.65 and 2.72 (2 br s, 3H total), 2.28-2.37 (m, 1H), 2.09-2.17 (m, 1H) , 1.74-1.90 (br m, 1H), 1.41-1.51 (m, 1H), 1.39 (s, 9H), 0.92-1.01 (m, 1H), 0.77-0.92 (m, 6H).
[0791] [0791] Step 4. Synthesis of tert-butyl (3R, 4S, 5S) -4 - {[(benzyloxy) carbonyl] (methyl) amino} -3-methoxy-5-methylheptanoate (# @ 2). To a solution of # 3 (8.4 g, 22 mmol, 1 eq.) In 1,2-dichloroethane (25 mL, 0.88 M) was added molecular sieves (4 Å, 0.7 g) and a sponge. Proton (1,8-bis (dimethylamino) naphthalene) (13.4 g, 59.2 mmol, 2.7 eq.), Followed by trimethyloxonium tetrafluoroborate (9.10 g, 61.6 mmol, 2.8 eq. ).
[0792] [0792] Step 5. Synthesis of tert-butyl (3R, 4S, 5S) -3-methoxy-5-methyl-4- (methylamino) heptanoate, hydrochloride salt (# 4). To a solution of # @ 2 (13.37 g, 33.98 mmol, 1 eq.) In methanol (134 mL, 0.1 M) and concentrated hydrochloric acid (3.1 mL, 37.4 mmol, 1, 1 eq.) 10% palladium on carbon (50% wet) (0.1% by weight; 1.34 g, 3.40 mmol) was added. The mixture was hydrogenated at 45 psi for 3 hours, then purged with nitrogen, filtered through Celite and concentrated in vacuo to provide # 4 (9.20 g, 92%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 9.65 (br s, 1H), 8.97 (br s, 1H), 3.98-4.04 (m, 1H), 3.40 (s, 3H) , 3.06-3.13 (br m, 1H), 2.82 (br dd, J = 6.5 Hz, 3H), 2.74-2.80 (m, 1H), 2.68 (dd , half of the ABX standard, J = 16.3, 4.2 Hz, 1H), 2.00-2.10 (br m, 1H), 1.73-1.84 (m, 1H), 1, 46 (s, 9H), 1.38-1.45 (m, 1H), 1.13 (d, J = 7.0 Hz, 3H), 0.99 (t, J = 7.4 Hz, 3H ).
[0793] [0793] Step 6. Synthesis of tert-butyl (3R, 4S, 5S) -4 - [{N - [(9H-fluoren-9-ylmethoxy) carbonyl] -L-valyl} (methyl) amino] -3- methoxy-5-methylheptanoate (# 5). To a mixture of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -L-valine (18.53 g, 54.60 mmol, 1.3 eq.) And 2-chloro-4,6-dimethoxy- 1,3,5-triazine (CDMT) (9.58 g, 54.6 mmol, 1.3 eq.) In 2-methyltetrahydrofuran (118.00 mL, 0.34 M) was added N-methylmorpholine (6, 52 mL, 59.1 mmol, 1.5 eq.) Followed by # 4 (11.80 g, 39.9 mmol, 1 eq.). After 3 hours, the reaction was quenched with water (50 ml) and stirred for 15 minutes. The aqueous layer was separated and re-extracted with 2-methyltetrahydrofuran (50 ml). The combined organic layers were washed with saturated aqueous sodium bicarbonate solution (50 ml), dried over magnesium sulfate, filtered, and concentrated in vacuo to provide a colorless oil, which was purified by silica gel chromatography (Gradient: 5 % to 40% ethyl acetate in heptane) to provide # 5 (26.2 g, 91%) as a colorless foam. LC-MS (Protocol I) m / z 581.3 [M + H +] 604.3 [M + Na +], retention time = 4.993 minutes; 1 H NMR (400 MHz, DMSO-d6), possibly in a mixture of rotamers, main characteristic signals: δ 7.88 (d, J = 7.4 Hz, 2H), 7.71 (d, J = 7, 4 Hz, 2H), 7.62 (d, J = 8.6 Hz, 1H), 7.41 (dd, J = 7.4, 7.4 Hz, 2H), 7.27-7.34 ( m, 2H), 4.13-4.32 (m, 4H), 3.70-3.82 (br m, 1H), 3.24 (s, 3H), 2.92 (br s, 3H) , 2.54 (dd, J = 15.7, 2.4 Hz, 1H), 2.17 (dd, J = 15.4, 9.4 Hz, 1H), 1.95-2.07 (m , 1H), 1.70-1.83 (br m, 1H), 1.40 (s, 9H), 0.83-0.94 (m, 9H), 0.69 (t, J = 7, 2 Hz, 3H).
[0794] [0794] Step 7A. Syntheses of tert-butyl (3R, 4S, 5S) -3-methoxy-5-methyl-4- [methyl- (L-valyl) -amino] -heptanoate (# 6). To a solution of # 5 (26 g, 42 mmol, 1 eq.) In tetrahydrofuran (260 ml, 0.16 M) was added diethylamine (22 ml) over 30 minutes. The reaction was stirred for about 6 hours and the suspension was then filtered through Celite and washed with additional tetrahydrofuran (25 ml). The filtrate was concentrated in vacuo to provide a pale yellow oil, which was again dissolved in 2-methyltetrahydrofuran (50 ml) and concentrated again to ensure complete removal of diethylamine. The crude oil of # 6 (> 15.25 g) was carried on to the next step without further purification.
[0795] [0795] Step 7B. Syntheses of (3R, 4S, 5S) -4 - [{N - [(9H-fluoren-9-ylmethoxy) carbonyl] -L-valyl} (methyl) amino] -3-methoxy-5-methylheptanoic (# @ 5). According to general procedure B, from # 5 (1.62 g, 2.79 mmol, 1 eq.), Dichloromethane (10 mL, 0.3 M) and trifluoroacetic acid (3 mL) were synthesized # @ 5 (1.42 g, 97%) as a solid that was used without further purification. LC-MS m / z 525.3 [M + H +] 547.3 [M + Na +] retention time = 0.95 minutes; 1H NMR (400 MHz, DMSO-d6), characteristic signals: δ 7.89 (d, J = 7.6 Hz, 2H), 7.71 (d, J = 7.4 Hz, 2H), 7.59 (d, J = 8.8 Hz, 1H), 7.41 (dd, J = 7.6, 7.4 Hz, 2H), 7.28-7.34 (m, 2H), 4.14- 4.32 (m, 4H), 3.24 (s, 3H), 2.92 (br s, 3H), 2.51 2.57 (m, 1H, assumed; partially obscured by the solvent peak), 2 , 20 (dd, J = 15.9, 9.5 Hz, 1H), 1.95-2.06 (m, 1H), 1.70-1.83 (br m, 1H),
[0796] [0796] Step 8. Syntheses of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -N-methyl-L-valyl-N- [(3R, 4S, 5S) -1-tert-butoxy-3- methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 7). To a mixture of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -N-methyl-L-valine (19.54 g, 55.29 mmol, 1.3 eq.) And # 6 (15.25 g, 42.54 mmol, 1 eq.) in 2-methyltetrahydrofuran (152 mL, 0.28 M) 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) (9.71 g, 55.3 mmol, 1.3 eq.). After 10 minutes, N-methylmorpholine (6.6 mL, 60 mmol, 1.4 eq.) Was added slowly, keeping the internal temperature below 25 ° C. The reaction was stirred for 4 hours and was then quenched by the addition of water (50 ml). After stirring for 15 minutes, the aqueous layer was separated and re-extracted with 2-methyltetrahydrofuran (50 ml). The combined organic layers were washed with saturated aqueous sodium bicarbonate solution (100 ml), then dried over magnesium sulfate, filtered, and concentrated in vacuo. The resulting yellow foam was purified by chromatography on silica gel (Gradient: 5% to 35% ethyl acetate in heptane) to provide # 7 (32 g, 97%). 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ 7.89 (d, J = 7.4 Hz, 2H), 7.62 (d, J = 7, 4 Hz, 2H), 7.41 (br dd,
[0797] [0797] Step 9. Synthesis of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -N-methyl-L-valyl-N- [(2R, 3S, 4S) -1-carboxy-2-methoxy- 4-methylhexan-3-yl] -N-methyl-L-valinamide (# 8). For # 7 (32 g, 46 mmol, 1 eq.) In dichloromethane (160 mL, 0.29 M) trifluoroacetic acid (17.4 mL, 231 mmol, 5 eq.) Was added dropwise over 10 minutes. After 6 hours, the same amount of trifluoroacetic acid was added and the reaction was continued for 18 hours. The reaction mixture was diluted with toluene (320 ml) and concentrated in vacuo to provide # 8 (35.8 g, 97%) as a pink oil, which was used in the next step without further purification. 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ 7.90 (d, J = 7.0 Hz, 2H), 7.62 (d, J = 7, 4 Hz, 2H), 7.41 (br dd, J = 7.4, 7.0 Hz, 2H), 7.29-7.35 (m, 2H), 4.54-4.68 (br m , 1H), [4.09 (d, J = 11 Hz) and 4.22 (d, J = 10.9 Hz), total 1H], 3.74-3.84 (br m, 1H), 3 , 22 and 3.24 (2 br s, 3H total), 2.94 and 2.96 (2 br s, 3H total), 2.78 and 2.80 (2 br s, 3H total), 2.13 -2.24 (m, 1H), 1.89-2.10 (br m, 2H), 1.70-1.81 (br m, 1H).
[0798] [0798] Preparation of (2R, 3R) -3 - [(2S) -1- (tert-butoxycarbonyl) pyrrolidin-2-yl] -3-methoxy-2-methylpropanoic (# 11; “Boc-Dap-acid ”)
[0799] [0799] Step 1. Synthesis of tert-butyl (2S) -2 - [(1R, 2S) -1-hydroxy-2-methylbut-3-en-1-yl] pyrrolidine-1-carboxylate (# 9). To a solution of tert-butyl (2S) -2-formylpyrrolidine-1-carboxylate (10 g, 50 mmol, 1 eq.) In dichloromethane (120 mL, 0.42 M) was added potassium (2Z) -2-buten -1-iltrifluoroborate (9.76 g, 60.2 mmol, 1.2 eq.) Followed by tetra-n-butylammonium bromide (3.24 g, 5.02 mmol, 0.1 eq.) And water ( 60 mL). After 13 hours, the reaction was diluted with dichloromethane (150 ml) and water (150 ml). The aqueous layer was separated and re-extracted with dichloromethane (100 ml). The combined organic layers were washed with aqueous sodium chloride solution (5 wt.%, 200 ml), washed with water (200 ml), and concentrated in vacuo to provide # 9 (~ 13 g) as an orange oil, which was used without further purification. 1 H NMR (400 MHz, CDCl3) δ 5.61 - 5.86 (br m, 1H), 4.97-5.09 (m, 2H), 3.80-3.98 (br m, 2H) , 3.45-3.67 (br m, 1H), 3.21 3.29 (m, 1H), 2.14-2.26 (m, 1H), 1.80-2.04 (m , 3H), 1.65-1.76 (m, 1H), 1.47 (s, 9H), 1.12 (d, J = 6.6 Hz, 3H).
[0800] [0800] Step 2. Synthesis of tert-butyl (2S) -2 - [(1R, 2S) -1-methoxy-2-methylbut-3-en-1-yl] pyrrolidine-1-carboxylate (# 10). Sodium hydride (60% in mineral oil, 3.38 g, 84.4 mmol, 1.1 eq.) Was combined with hexane (40 mL), and the mixture was subjected to rapid mechanical stirring for 5 minutes. The solids were allowed to settle and the hexane was removed. This procedure was repeated twice to remove mineral oil. N, N-dimethylformamide (59 ml, 1.3 M) was added and the mixture was cooled to 0 ° C; methyl iodide (5 mL; 81 mmol, 1.05 eq.) was then added dropwise, followed by the dropwise addition of a solution of # 9 (19.6 g, 76.8 mmol, 1 eq. .) in N, N-dimethylformamide (59 mL) over 5 minutes, maintaining the temperature between 0 ° C and 5 ° C. The reaction was stirred at 0 ° C for 2 hours. The reaction was quenched with saturated aqueous ammonium chloride solution (150 mL), poured into aqueous sodium chloride solution (5% by weight, 300 mL), and the mixture was extracted with ethyl acetate (3 x 50 mL) . The combined organic layers were washed with 10% aqueous sodium chloride solution (2 x 300 ml), washed with water (200 ml), and concentrated again in vacuo. The resulting oil-in-water oil was reconcentrated from ethyl acetate (150 mL) and purified by chromatography on silica gel (Gradient: 2% to 10% ethyl acetate in heptane) to provide # 10 (15.0 g, 73% ) as a colorless oil. 1H NMR (400 MHz, CDCl3),
[0801] [0801] Step 3. Synthesis of (2R, 3R) -3 - [(2S) -1- (tert-butoxycarbonyl) pyrrolidin-2-yl] -3-methoxy-2-methylpropanoic acid (# 11). For # 10 (25.0 g, 92.8 mmol, 1 eq.) In tert-butanol (100 mL, 0.93 M) water (30.00 mL) was added immediately, followed by N-methylmorpholine-N- oxide (25.97 g, 192.1 mmol, 2.07 eq.) and osmium tetroxide (235.93 mg, 928.04 µmol, 0.01 eq.). After 12 hours, the mixture was concentrated in vacuo using water (20 ml) to azeotropically remove residual tert-butanol. The residue was partitioned between ethyl acetate (500 ml) and water (500 ml) with brine (150 ml). The aqueous layer was re-extracted with ethyl acetate (250 ml). The combined organic layers were washed with aqueous sodium chloride solution (10% by weight, 200 ml), washed with water (150 ml), and concentrated in vacuo to provide a pale brown oil moist in water, which was reconcentrated from ethyl acetate (100 mL) to remove any remaining water. This crude diol (34.76 g) was used without further purification. For the crude diol (34.76 g, 92.8 mmol, 1 eq.) In acetonitrile (347 mL, 0.1 M) and water (174 mL), sodium permanganate (2.03 g, 5.73 mmol, 0.05 eq.). The mixture was cooled to 0 ° C and sodium periodate (51.46 g, 240.6 mmol, 2.1 eq.) Was added in portions over 30 minutes, keeping the internal temperature below 5 ° C. The reaction was stirred at 0 ° C for 4 hours and was then poured into a solution of sodium thiosulfate pentahydrate (65.40 g, 263.5 mmol, 2.3 eq.) In water (100 mL) . The mixture was filtered through Celite and the filtrate was concentrated in vacuo. The residue was partitioned between ethyl acetate (200 ml) and water (200 ml). The aqueous layer was re-extracted with ethyl acetate (250 ml), and the combined organic layers were washed with 10% aqueous citric acid solution. As the desired product was very soluble in water, all aqueous layers were combined, treated with Celite (100 g) and concentrated in vacuo, to give an off-white paste. Ethyl acetate (150 ml) was added and the mixture was reconcentrated to remove any residual water; this operation was repeated once more. The slurry was treated with ethyl acetate (150 ml) and placed in vacuo, at 50 ° C for 10 minutes and filtered (repeated twice). These filtrates were combined with the previous organic layer (from washing with citric acid), concentrated, diluted with ethyl acetate (200 ml) and filtered through Celite to remove the solids. Finally, this filtrate was concentrated to yield # 11 (22.9 g, 69% over two steps) as a yellowish / brown foam. LCMS (Protocol I): m / z 310.1 [M + Na +], 232.1 [(M-2-methylprop-1-ene) + H +], 188.1 [(M - Boc) + H +], retention time = 3.268 minutes; 1H NMR (400 MHz, DMSO-d6), characteristic signals: δ 3.61 - 3.85 (br m, 2H), 3.20-3.45 (br m, 4H), 3.03-3.17 (br m, 1H), 1.59-1.93 (br m, 4H), 1.40 (br s, 9H), 1.02-1.18 (br m, 3H).
[0802] [0802] Preparation of (2R, 3R) -3-Methoxy-2-methyl-N - [(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] - 3 - [( 2S) -pyrrolidin-2-yl] propanamide, trifluoroacetic acid salt (# 19) and (2R, 3R) -3-methoxy-2-methyl-N - [(1S) -2-phenyl-1- (1, 3-thiazol-2-yl) ethyl] -3 - [(2S) -pyrrolidin-2-yl] propanothioamide, trifluoroacetic acid salt (# 18) - $%. / '% ()% "* +, $ 0 /'% ()% $% 0 112 $% &&&&&&&&&&&&&&&&&&&&&&&&&&&
[0803] [0803] Step 1. Synthesis of N - (tert-butoxycarbonyl) -L-phenylalaninamide (# 12). To a solution of Boc-Phe-OH (30.1 g, 113 mmol, 1 eq.) In tetrahydrofuran (378 mL, 0.3 M) cooled to -10 ° C was added N-methylmorpholine (13.6 mL, 124 mmol , 1.09 eq.), And ethyl chloroformate (11.8 ml, 124 mmol, 1.09 eq.). After 20 minutes, 30% aqueous ammonium hydroxide solution (45 mL, 350 mmol, 3.1 eq.) Was added. The mixture was stirred at room temperature for 18 hours before being concentrated in vacuo. The residue was diluted with ethyl acetate and washed sequentially with 1 N aqueous potassium bisulfate solution, water and brine. The organic layer was then dried over sodium sulfate, filtered, and concentrated in vacuo. The white solid was dissolved (this heating required with stirring) in ethyl acetate (about 400 ml); the solution was then allowed to cool to room temperature before adding hexane (~ 1000 ml). After a few minutes, a white material started to precipitate from the reaction mixture. The solid was collected by filtration, washed with heptane (2 x ~ 150 ml), and dried in vacuo for 18 hours to provide # 12 (24.50 g, 82%) as a solid. LC-MS: m / z 263.2 [M-H +], 309.2 [M + HCO2-], retention time = 1.85 minutes; 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, main rotamer: δ 7.35 (br s, 1H), 7.22-7.30 (m, 5H), 7.00 (br s, 1H), 6.78 (d, J = 8.6 Hz, 1H), 4.09 (ddd, J = 10, 9, 4.5 Hz, 1H), 2.95 (dd, J = 13.8, 4.4 Hz, 1H), 2.72 (dd, J = 13.7, 10.1 Hz, 1H), 1.30 (s, 9H).
[0804] [0804] Step 2. Synthesis of tert-butyl - [(2S) -1-amino-3-phenyl-1-thioxopropan-2-yl] carbamate (# 13). To a solution of # 12 (14.060 g, 53.192 mmol, 1 eq.) In tetrahydrofuran (180 mL, 0.296 M), was added 2,4-bis (4-methoxyphenyl) -1,3,2,4-dithiadiphosphethane- 2,4-dithione (Lawesson's reagent) (12.70 g, 31.40 mmol, 0.59 eq.) And the reaction was refluxed for 90 minutes. The reaction was cooled to room temperature and quenched by the addition of saturated aqueous sodium bicarbonate solution. The mixture was extracted twice with ethyl acetate and the combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was dissolved in ethyl acetate, concentrated in vacuo over silica and purified by silica gel chromatography (Gradient: 0% to 100% ethyl acetate in heptane), providing # 13 (11.50 g, 77%) as a solid White. LC-MS: m / z 279.4 [M-H +], 225.2 [(M - 2-methyl-prop-1-ene) + H +], 181.2 [(M - Boc) + H +]; 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, main rotamer: δ 9.60 (br s, 1H), 9.19 (br s, 1H), 7.23-7, 32 (m, 5H), 6.82 (d, J = 8.8 Hz, 1H), 4.44 (ddd, J = 9.4, 9.1, 4.4 Hz, 1H), 3.00 (dd, J = 13.7, 4.5 Hz, 1H), 2.79 (dd, J = 13.6, 9.9 Hz, 1H), 1.29 (s, 9H).
[0805] [0805] Step 3. Synthesis of tert-butyl - [(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] carbamate (# 14). To a mixture of # 13 (5.65 g, 20.2 mmol, 1 eq.) In acetone (101 mL, 0.2 M) was added diethyl acetal bromoacetaldehyde (8.76 mL, 58.2 mmol, 2, 89 eq.) And 2 drops of 4 M hydrochloric acid in dioxane. The mixture was degassed with nitrogen, three times, before being heated to reflux. After 2 hours, the reaction was cooled to room temperature and concentrated in vacuo. The residue was dissolved in ethyl acetate, washed with saturated aqueous sodium bicarbonate solution and washed with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting crude orange oil was diluted with ethyl acetate before being concentrated in vacuo on silica and purified by chromatography on silica gel (Gradient: 0% to 35% ethyl acetate in heptane) and then by reverse phase chromatography ( Method A), to provide # 14 (625 mg, 10%); HPLC (Protocol E): m / z 304.5 [M + H +], 248.9 [(M - 2-methyl-prop-1-ene) + H +], retention time = 7.416 minutes; 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, main rotamer: δ 7.75 (d, J = 3.3 Hz, 1H), 7.75 (br d, J = 8 , 6 Hz, 1H), 7.61 (br d, J = 3.1 Hz, 1H), 7.25-7.30 (m, 5H), 4.99 (ddd, J = 10.5, 8 , 9, 4.5 Hz, 1H), 3.29-3.36 (m, 1H, assumed; partially obscured by water signal), 2.98 (dd, J = 13.8, 10.6 Hz, 1H), 1.31 (s, 9H).
[0806] [0806] Step 4. Syntheses of (1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethanamine, hydrochloride salt (# 15). According to general procedure C, from # 14 (1.010 g, 3.318 mmol, 1 eq.), Dioxane (10 mL, 0.33 M) and a 4 M hydrochloric acid solution in dioxane (20 mL, 80 mmol, 20 eq.) # 15 (775 mg, 97%) were synthesized. 1H NMR (400 MHz, DMSO-d6) δ 8.95-9.07 (br m, 3H), 7.86 (d, J = 3.2 Hz, 1H), 7.73 (d, J = 3 , 2 Hz, 1H), 7.18-7.28 (m, 3H), 7.10-7.15 (m, 2H), 4.98-5.07 (m, 1H), 3.49 ( dd, J = 13.3, 4.9 Hz, 1H), 3.18 (dd, J = 13.4, 10.2 Hz, 1H).
[0807] [0807] Step 5. Synthesis of tert-butyl (2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3-oxo-3- {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidine-1-carboxylate (# 16). To a solution of # 11 (280 mg, 0.974 mmol, 1 eq.) And # 15 (460 mg, 1.44 mmol, 1.48 eq.) In N, N-dimethylformamide (3 mL, 0.32 M) at 0 ° C diethylphosphoryl cyanide (DEPC) (93% pure, 212 µL, 1.30 mmol, 1.34 eq.) was added, followed by triethylamine (367 µL, 2.63 mmol, 2.7 eq. ). After 2 hours at 0 ° C, the reaction mixture was heated to room temperature for 18 hours. The reaction mixture was then diluted with ethyl acetate: toluene (2: 1, 30 mL) and washed successively with 1 M aqueous sodium bisulfate solution (35 mL) and 50% sodium bicarbonate solution. saturated aqueous sodium (4 x 25 mL). The organic layer was dried over sodium sulfate, filtered, concentrated in vacuo, and purified by chromatography on silica gel (12% to 100% ethyl acetate in heptane) to provide # 16 as a light amber oil (374 mg, 81% ). LC-MS: m / z 474.4 [M + H +], 374.4 [(M - 2-methyl-prop-1-ene) + H +] Retention time = 3.63 minutes; 1H NMR (400 MHz, DMSO-d6), characteristic signals: δ 8.66 (d, J = 8.5 Hz, 1H), 7.78 (d, J = 3.3 Hz, 1H), 7.64 (d, J = 3.3 Hz, 1H), 7.21-7.31 (m, 4H), 7.14-7.20 (m, 1H), 5.40 (ddd, J = 11.4 , 8.5, 4.0 Hz, 1H), 3.23 (br s, 3H), 2.18 (dq, J = 9.7, 6.7 Hz, 1H), 1.06 (d, J = 6.6 Hz, 3H).
[0808] [0808] Step 6A. Syntheses of tert-butyl (2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl ) ethyl] amino} -3-thioxopropyl] pyrrolidine-1-carboxylate (# 17). A mixture of # 16 (350 mg, 0.739 mmol, 1 eq.) And 2,4-bis (4-methoxyphenyl) -1,3,2,4-
[0809] [0809] Step 6B. Syntheses of (2R, 3R) -3-methoxy-2-methyl-N - [(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] -3 - [(2S) - pyrrolidin-2-yl] propanetioamide, trifluoroacetic acid salt (# 18). According to general procedure B, from # 17 (198 mg, 0.404 mmol, 1 eq.), Dichloromethane (6 ml, 0.07 M) and trifluoroacetic acid (2 ml) were synthesized # 18 (185 mg, 91%), which were used without further purification. LC-MS: m / z 390.1 [M + H +], retention time = 0.57 minutes; 1H NMR (400 MHz, DMSO-d6) δ 10.91 (d, J = 8.2 Hz, 1H), 9.07-9.20 (br m, 1H), 7.86-8.00 (br m, 1H), 7.83 (d, J = 3.2 Hz, 1H), 7.69 (d, J = 3.3 Hz, 1H), 7.27-7.36 (m, 4H), 7.21-7.26 (m, 1H), 6.33 (ddd, J = 11.3, 8.3, 4.4 Hz, 1H), 3.76-3.82 (m, 1H), 3.56 (dd, J = 14.6, 4.3 Hz, 1H), 3.45 (s, 3H), 3.28 (dd, J = 14.6, 11.3 Hz, 1H), 3 , 02-3.12 (br m, 1H), 2.89-3.00 (br m, 1H), 2.72-2.89 (m, 2H), 1.69-1.83 (br m , 1H), 1.43-1.58 (m, 2H), 1.20-1.33 (m, 1H), 1.22 (d, J = 6.6 Hz, 3H).
[0810] [0810] Step 7. Synthesis of (2R, 3R) -3-methoxy-2-methyl-N - [(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] -3 - [(2S) -pyrrolidin-2-yl] propanamide, trifluoroacetic acid salt (# 19). According to general procedure B, starting at # 16 (607 mg, 1.28 mmol, 1 eq.),
[0811] [0811] Preparation of (2R, 3R) -3-methoxy-2-methyl-N- (2-phenylethyl) -3 - [(2S) - pyrrolidin-2-yl] propanothioamide, hydrochloride salt (# 23) and (2R, 3R) -3-methoxy-2-methyl-N- (2-phenylethyl) -3 - [(2S) -pyrrolidin-2-yl] propanamide, hydrochloride salt (# 24) $% &% 2 $% 3 & +%
[0812] [0812] Step 1A. Synthesis of tert-butyl (2S) -2 - {(1R, 2R) -1-methoxy-2-methyl-3-oxo-3- [(2-phenylethyl) amino] propyl} -pyrrolidine-1-carboxylate (# 20). For # 11 (22 g, 77 mmol, 1 eq.) In dichloromethane (383 ml, 0.2 M) and N, N-dimethylformamide (30 ml), diisopropylethylamine (26.9 ml, 153 mmol, 2 eq. ), 2-phenylethylamine (11.6 ml, 91.9 mmol, 1.2 eq.) And HATU (39.0 g, 99.5 mmol, 1.3 eq.). The reaction was stirred for 18 hours and then concentrated in vacuo. The residue was taken up in ethyl acetate (700 ml) and washed, sequentially, with 1 M aqueous hydrochloric acid solution (2 x 200 ml) and brine. The organic layer was dried over sodium sulfate, filtered and evaporated in vacuo. The crude material was taken up in dichloromethane and filtered. The filtrate was purified by silica gel chromatography (Gradient: 0% to 100% ethyl acetate in heptane) to provide # 20 (24 g, 80%) as an off-white solid. LC-MS: m / z 392.2 [M +2 H +], 291.1 [(M - Boc) + H +], retention time = 0.88 minutes; 1H NMR (400 MHz, DMSO-d6), it is assumed to be a mixture of rotamers: δ 7.80-7.89 (br m, 1H), 7.23-7.29 (m, 2H), 7, 15-7.23 (m, 3H), 3.72-3.82 and 3.55-3.62 (2 br m, 1H total), 3.45-3.55 (br m, 1H), 3 , 31-3.44 (br m, 2H), 3.29 (s, 3H), 3.12-3.25 (br m, 1H), 2.98-3.12 (br m, 1H), 2.71 (t, J = 7.1 Hz, 2H), 2.09-2.19 (m, 1H), 1.71-1.83 (br m, 2H), 1.60-1.70 (br m, 1H), 1.49-1.60 (br m, 1H), 1.41 (s, 9H), 1.03 (d, J = 6.8 Hz, 3H).
[0813] [0813] Step 1B. Syntheses of dipyridinium-1-ylpentatiodiphosphonate (# 21).
[0814] [0814] Step 2A. Synthesis of tert-butyl (2S) -2 - {(1R, 2R) -1-methoxy-2-methyl-3 - [(2-phenylethyl) amino] -3-thioxopropyl} pyrrolidine-1-carboxylate (# 22) . A mixture of # 20 (1,200 g, 3,073 mmol, 1 eq.) And # 21 (1.40 g, 3.69 mmol, 1.2 eq.) In acetonitrile (15 mL, 0.20 M) was subjected to microwave radiation at 100 ° C for 30 minutes. The reaction mixture was then cooled to room temperature, diluted with ethyl acetate (150 ml), and washed sequentially with 0.5 M aqueous hydrochloric acid solution (100 ml) and brine (2 x 50 ml) . The organic layer was dried over magnesium sulfate, filtered, and concentrated in vacuo. The residue was purified by chromatography on silica gel (Gradient: 20% to 80% ethyl acetate in heptane) to provide # 22 (670 mg, 54%) as a white wax-like solid; mp: 107-
[0815] [0815] Step 3. Synthesis of (2R, 3R) -3-methoxy-2-methyl-N- (2-phenylethyl) -3 - [(2S) - pyrrolidin-2-yl] propanothioamide, hydrochloride salt (# 23). According to procedure C, from # 22 (325 mg, 0.799 mmol, 1 eq.), Dioxane (5 mL, 0.2 M) and 4 M of a solution of hydrochloric acid in dioxane (4 mL, 16 mmol, 20 eq.) # 23 (274 mg, quantitative) was synthesized as a white foam; LC-MS: 308.2 [M + H +], retention time = 0.55 minutes.
[0816] [0816] Step 2B. Syntheses of (2R, 3R) -3-methoxy-2-methyl-N- (2-phenylethyl) -3 - [(2S) - pyrrolidin-2-yl] propanamide, hydrochloride salt (# 24). To # 20 (7.00 g, 17.9 mmol, 1 eq.) In dioxane (50 ml, 0.36 M) and methanol (2 ml) were added 4 M of a solution of hydrochloric acid in dioxane (20 mL, 80 mmol, 4.4 eq.). After stirring for 18 hours, the mixture was concentrated to provide # 24 (5.86 g, quantitative) as a gum, which was used without further purification; LC-MS: 292.2 [M + H +], retention time = 0.47 minutes.
[0817] [0817] Preparation of N-methyl-L-valyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1- methoxy-2- methyl-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} -3-thioxopropyl] pyrrolidin-1-yl} - 5-methyl-1-oxoheptan -4-yl] -N-methyl-L-valinamide (# 26)
[0818] [0818] Step 1. Syntheses of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -N-methyl-L-valyl-N- [(3R, 4S, 5S) -3-methoxy-1 - {( 2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} - 3-thioxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 25)
[0819] [0819] According to general procedure D, from # 8 (480 mg, 0.753 mmol, 1 eq.), Dichloromethane (10 mL, 0.07 M), N, N-dimethylformamide (2 mL), amine # 18 (401 mg, 0.941 mmol, 1.25 eq.), HATU (372 mg, 0.979 mmol, 1.3 eq.) and triethylamine (367 µL, 2.64 mmol, 3.5 eq.), was The desired crude material was synthesized, which was purified by chromatography on silica gel (Gradient: 0% to 30% acetone in heptane) to provide # 25 (711 mg, 75%) as a solid. LC-MS: m / z 1009.7 [M + H +], retention time = 1.15 minutes; HPLC (Protocol B): m / z 505.3 [M +2 H +] / 2, retention time = 10.138 minutes; 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ [10.54 (br d, J = 8 Hz) and 10.81 (br d, J = 8 Hz) , Total 1H], 7.89 (br d, J = 7 Hz, 2H), [7.80 (d, J = 3.3 Hz) and 7.83 (d, J = 3.2 Hz), 1H total], [7.64 (d, J = 3.2 Hz) and 7.69 (d, J = 3.2 Hz), total 1H], 7.62 (br d, J = 7 Hz, 2H) , 7.37-7.44 (m, 2H), 7.28-7.35 (m, 4H), 7.20-7.27 (m, 2H), 7.12-7.18 (m, 1H), 6.27-6.35 and 6.40-6.48 (2 m, 1H total), [1.14 (d, J = 6.4 Hz) and 1.17 (d, J = 6 , 3 Hz), total 3H].
[0820] [0820] Step 2. Synthesis of N-methyl-L-valyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2- [(1R, 2R) -1-methoxy -2-methyl-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} -3-thioxopropyl] pyrrolidin-1-yl} -5-methyl- 1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 26). According to general procedure A, from # 25 (701 mg, 0.694 mmol) in dichloromethane (10 mL, 0.07 M) and diethylamine (10 mL), the desired crude material was synthesized, which was purified by chromatography on silica gel (Gradient: 0% to 10% methanol in dichloromethane) to provide a glassy solid. Diethyl ether and heptane were added and the mixture was concentrated in vacuo, yielding # 26 (501 mg, 92%) as a white solid. HPLC (Protocol A): m / z 787.4 [M + H +], retention time = 7.229 minutes (purity> 97%); 1H NMR (400 MHz, DMSO-d6), is presumed to be a mixture of rotamers, characteristic signals: δ [10.54 (br d, J = 8 Hz) and 10.81 (br d, J = 8 Hz. ), 1H total], [7.99 (br d, J = 9 Hz) and 8.00 (br d, J = 9 Hz), 1H total], [7.80 (d, J = 3.3 Hz) ) and 7.83 (d, J = 3.3 Hz), total 1H], [7.65 (d, J = 3.2 Hz) and 7.69 (d, J = 3.3 Hz), 1H total], 7.29-7.34 (m, 2H), 7.19-7.28 (m, 2H), 7.13-7.19 (m, 1H), [6.31 (ddd, J = 11, 8, 4.5 Hz) and 6.45 (ddd, J = 11.5, 8, 4.5 Hz), 1H total], [4.57 (dd, J = 8.9, 8, 7 Hz) and 4.63 (dd, J = 8.7, 8.7 Hz), 1H total], 3.16, 3.21, 3.24 and 3.25 (4 s, 6H total), 2 , 96 and 3.03 (2 br s, 3H total), [1.14 (d, J = 6.6 Hz) and 1.17 (d, J = 6.4 Hz), 3H total].
[0821] [0821] Preparation of N2 - [(1-Aminocyclopentyl) carbonyl] -N - [(3R, 4S, 5S) -3-methoxy-1- {(2S) -2 - [(1R, 2R) -1-methoxy -2-methyl-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} -3-thioxopropyl] pyrrolidin-1-yl} -5-methyl- 1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 30) 0 &. * &% /%% $
[0822] [0822] Step 1. Synthesis of tert-butyl (3R, 4S, 5S) -4 - [{N - [(1 - {[(9H-fluoren-9-ylmethoxy) carbonyl] amino} -cyclopentyl) carbonyl] - L-valyl} (methyl) amino] -3-methoxy-5-
[0823] [0823] Step 2. (3R, 4S, 5S) -4 - [{N - [(1 - {[(9H-fluoren-9-ylmethoxy) carbonyl] amino} -cyclopentyl) carbonyl] -L- valyl} (methyl) amino] -3-methoxy-5-methylheptanoic (# 28). To a solution of # 27 (500 mg, 0.723 mmol) in dichloromethane (7 ml, 0.1 M) was added trifluoroacetic acid (3 ml). The reaction mixture initially turned orange, then darkened over time. After stirring for 18 hours, the solvent was removed in vacuo to provide # 28 (460 mg, quantitative) as a dark brown glass, which was used without further purification. LC-MS: m / z 636.3 [M + H +].
[0824] [0824] Step 3. Synthesis of N2 - [(1 - {[(9H-fluoren-9-ylmethoxy) carbonyl] amino} - cyclopentyl) carbonyl] -N - [(3R, 4S, 5S) -3-methoxy- 1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3- {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl ] amino} -3-thioxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 29). According to general procedure D,
[0825] [0825] Step 4. Synthesis of N2 - [(1-aminocyclopentyl) carbonyl] -N - [(3R, 4S, 5S) -3- methoxy-1 - {(2S) -2 - [(1R, 2R) - 1-methoxy-2-methyl-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} -3-thioxopropyl] pyrrolidin-1-yl} -5 -methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 30). According to general procedure A, from # 29 (54 mg, 0.054 mmol) in dichloromethane (6 ml, 0.9 mM) and diethylamine (4 ml), the desired crude material was synthesized, which was purified by chromatography on silica gel (Gradient: 0% to 10% methanol in dichloromethane) to provide Example # 30 (26 mg, 61%) as a solid. HPLC (protocol A): retention time = 7.233 minutes, m / z 785.4 [M + H +], (purity> 72%). 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ [10.54 (br d, J = 8 Hz) and 10.82 (br d, J = 8 Hz) , Total 1H], 8.19-8.27 (m, 1H), [7.80 (d, J = 3.2 Hz) and 7.83 (d, J = 3.2 Hz), total 1H] , [7.65 (d, J = 3.3 Hz) and 7.69 (d, J = 3.3 Hz), total 1H], 7.28-7.33 (m, 2H), 7.20 -7.27 (m, 2H), 7.14-7.19 (m, 1H), [6.31 (ddd, J = 11, 8, 4.5 Hz) and 6.44 (ddd, J = 11, 8, 4 Hz), 1H total], [4.53 (dd, J = 9, 8 Hz) and 4.60 (dd, J = 9,
[0826] [0826] Preparation of 2-Methylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1- methoxy-2-methyl-3- {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} -3-thioxopropyl] pyrrolidin-1-yl} - 5-methyl-1-oxoheptan-4-yl ] -N-methyl-L-valinamide (# 34) 4 & * & 5 &%,
[0827] [0827] Step 1. Syntheses of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -2-methylalanyl-N- [(3R, 4S, 5S) -1-tert-butoxy-3-methoxy-5- methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 31). To a solution of # 6 (70% pure, 3.13 g, 6.1 mmol, 1 eq.) In dichloromethane (40 mL, 0.15 M) was added N - [(9H-fluoren-9-ylmethoxy) carbonyl] -2-methylalanine (1.99 g, 6.12 mmol, 1 eq.), diisopropylethylamine (2.67 mL, 15.3 mmol, 2.5 eq.) and HATU (2.79 g, 7, 35 mmol, 1.2 eq.). The reaction mixture was stirred for 18 hours, diluted with ethyl acetate, washed with 1 M aqueous hydrochloric acid solution and washed with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo over silica. The material was then purified by chromatography on silica gel (Gradient: 0% to 45% ethyl acetate in heptane) to provide # 31 (3.65 g, 90%) as a solid. LC-MS: m / z 665.5 [M + H +], 688.5 [M + Na +], 610.5 [(M - 2-methyl-prop-1-ene) + H +]; HPLC (Protocol C): retention time = 9.455 (purity> 94%); 1H NMR (400 MHz, DMSO-d6), characteristic signals: δ 7.89 (d, J = 7.4 Hz, 2H), 7.67-7.74 (m, 2H), 7.39-7, 48 (m, 3H), 7.31-7.36 (m, 2H), 7.29 (br d, J = 8.8 Hz, 1H), 4.47-4.60 (br m, 1H) , 4.47 (dd, J = 8.6, 8.0 Hz, 1H), 4.18-4.28 (m,
[0828] [0828] Step 2. Syntheses of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -2-methylalanyl-N- [(2R, 3S, 4S) -1-carboxy-2-methoxy-4-methylhexan- 3-yl] -N-methyl-L-valinamide (# 32). According to general procedure B, starting from # 31 (500 mg, 0.751 mmol) in dichloromethane (7 mL, 0.1 M) and trifluoroacetic acid (3 mL) was synthesized # 32 as a glass (458 mg, quantitative ), which was used in the next step, without further purification. LC-MS: m / z 611.4 [M + 2 H +], 632.2 [M + Na +], retention time = 0.94 minutes.
[0829] [0829] Step 3. Synthesis of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -2-methylalanyl-N- [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} -3-thioxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 33). According to general procedure D, from # 32 (53.0 mg, 0.083 mmol, 1 eq.), Dichloromethane (4 mL, 0.02 M), N, N-dimethylformamide (1 mL), amine # 18 (43.8 mg, 0.0870 mmol, 1 eq.), Triethylamine (36 µL, 0.26 mmol, 3 eq.) And HATU (39.5 mg, 0.104 mmol, 1.2 eq.), Was The desired crude material was synthesized, which was purified by chromatography on silica gel (Gradient: 0% to 30% acetone in heptane) to provide # 33 (60 mg, 69% for both steps). LC-MS: m / z 981.4 [M + H +], retention time = 1.090 minutes; 1H NMR (400 MHz, DMSO-d6), is presumed to be a mixture of rotamers, characteristic signals: δ [10.54 (br d, J = 8 Hz) and 10.80 (br d, J = 8 Hz) , Total 1H], 7.86-7.91 (m, 2H), [7.80 (d, J = 3.3 Hz) and 7.82 (d, J = 3.3 Hz), total 1H] , 7.68-7.74 (m, 2H), [7.64 (d, J = 3.2 Hz) and 7.68 (d, J = 3.3 Hz), total 1H], 7.38 -7.44 (m, 2H), 7.20-7.36 (m, 6H), 7.12-7.17 (m, 1H), 6.27-6.34 and 6.40 -6, 47 (2 m, 1H total), 3.22 and 3.24 (2 s, 3H total), 3.14 and 3.18 (2 s, 3H total), 2.90 and 2.97 (2 br s , 3H total), 1.37 (br s, 3H), 1.31 (2 br s, 3H total), [1.13 (d, J = 6.6 Hz) and 1.16 (d, J = 6.5 Hz), 3H total].
[0830] [0830] Step 4. Synthesis of 2-methylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2- [(1R, 2R) -1-methoxy-2-methyl -3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} -3-thioxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan- 4-yl] -N-methyl-L-valinamide (# 34). According to general procedure A, from # 33 (55 mg, 0.055 mmol, 1 eq.) In dichloromethane (6 ml, 0.009 M) and diethylamine (4 ml), the desired crude material was synthesized, which was purified by chromatography on silica gel (Gradient: 0% to 5% methanol in dichloromethane) to provide # 34 (25 mg, 60%) as a solid. HPLC (Protocol A): m / z 759.4 [M + H +], retention time = 7.088 minutes (purity> 75%). 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ [10.54 (br d, J = 8 Hz) and 10.81 (br d, J = 8 Hz) , Total 1H], 8.01-8.08 (m, 1H), [7.80 (d, J = 3.1 Hz) and 7.83 (d, J = 3.3 Hz), total 1H] , [7.65 (d, J = 3.2 Hz) and 7.69 (d, J = 3.2 Hz), total 1H], 7.29-7.33 (m, 2H), 7.20 -7.27 (m, 2H), 7.13- 7.19 (m, 1H), 6.27-6.35 and 6.40-6.48 (2 m, 1H total), [4.49 (dd, J = 9, 8 Hz) and 4.56 (dd, J = 9, 8 Hz), total 1H], 3.24 and 3.25 (2 s, 3H total), 3.17 and 3, 21 (2 s, 3H total), 2.92 and 2.99 (2 br s, 3H total), 1.20 and 1.21 (2 s, 3H total), 1.12 and 1.13 (2 s , 3H total), 0.75-0.81 (m, 3H).
[0831] [0831] Preparation of N-methyl-L-valyl-N - {(3R, 4S, 5S) -3-methoxy-1 - [(2S) -2 - {(1R, 2R) -1- methoxy-2- methyl-3 - [(2-phenylethyl) amino] -3-thioxopropyl} pyrrolidin-1-yl] -5-methyl-1-oxoheptan-4-yl} -N-methyl-L-valinamide (# 36) A " 7
[0832] [0832] Step 1. Syntheses of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -N-methyl-L-valyl-N- {(3R, 4S, 5S) -3-methoxy-1 - [( 2S) -2 - {(1R, 2R) -1-methoxy-2-methyl-3 - [(2-phenylethyl) amino] -3-thioxopropyl} pyrrolidin-1-yl] -5-methyl-1-oxoheptan- 4-yl} -N-methyl-L-valinamide (# 35). To a mixture of # 23 (337 mg, 0.983 mmol, 1 eq.) In dichloromethane (8 mL, 0.1 M) and N, N-
[0833] [0833] Step 2. Syntheses of N-methyl-L-valyl-N - {(3R, 4S, 5S) -3-methoxy-1 - [(2S) -2- {(1R, 2R) -1-methoxy -2-methyl-3 - [(2-phenylethyl) amino] -3-thioxopropyl} pyrrolidin-1-yl] -5-methyl-1-oxoheptan-4-yl} -N-methyl-L-valinamide (# 36 ). According to general procedure A, starting from # 35 (465 mg, 0.502 mmol, 1 eq.) In dichloromethane (5 ml, 0.1 M) and diethylamine (5 ml), the desired crude material was synthesized, which was purified by silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) to provide # 36 (310 mg, 88%) as a solid. LC-MS: m / z 704.6 [M + H +], retention time = 0.74 minutes; HRMS: m / z calculated for C38H66N5O5S: 704.4779, found: 704.477 [M + H +]; 1H NMR (400 MHz, CD3OD), presumed to be a mixture of rotamers, characteristic signals: δ 7.23-7.30 (m, 4H), 7.15-7.22 (m, 1H), [4 , 68 (d, J = 8.6 Hz) and 4.74 (d, J = 8.0 Hz), 1H total], 3.39 and 3.40 (2 s, 3H total), 3.12 and 3.22 (2 br s, 3H total), [2.82 (d, J = 6.0 Hz) and 2.84 (d, J = 6.0 Hz), 1H total], 2.29 and 2 , 30 (2 s, 3H total), [1.27 (d, J = 6.8 Hz) and 1.29 (d, J = 6.6 Hz), 3H total], [0.84 (t, J = 7.4 Hz) and 0.87 (t, J = 7.4 Hz), total 3H].
[0834] [0834] Preparation of N-methyl-L-valyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1- carboxy-2-phenylethyl] amino} -1-methoxy-2-methyl-3-thioxopropyl] pyrrolidin-1-yl} -3-methoxy-5-
[0835] [0835] Step 1. Methyl syntheses N - {(2R, 3R) -3 - [(2S) -1- (tert-butoxycarbonyl) pyrrolidin-2-yl] -3-methoxy-2-methylpropanoyl} -L- phenylalaninate (# 37). To a mixture of # 11 (2.7 g, 9.4 mmol, 1 eq.) In dichloromethane (30 ml, 0.3 M) and N, N-dimethylformamide (3 ml) were added diisopropylethylamine (3.30 ml , 18.8 mmol, 2 eq.), L-phenylalanine methyl ester hydrochloride (2.03 g, 9.40 mmol, 1.2 eq.) And HATU (4.79 g, 12.2 mmol, 1.3 eq.). The reaction was stirred for 18 hours and then concentrated in vacuo. The residue was taken up in ethyl acetate (100 ml) and washed sequentially with 1 M hydrochloric acid (2 x 50 ml) and brine. The organic layer was dried over sodium sulfate, filtered and evaporated in vacuo. The crude material was taken up in dichloromethane and filtered. The filtrate was purified by chromatography on silica gel (Gradient: 0% to 100% ethyl acetate in heptane) to provide # 37 (2.76 g, 65%) as an off-white solid. LC-MS: m / z 449.3 [M + H +], 349.2 [(M - Boc) + H +] retention time = 0.88 minutes; 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ 8.28 (d, J = 8.2 Hz, 1H), 7.14-7.29 (m, 5H), 4.50 (ddd, J = 10.9, 8.1, 4.4 Hz, 1H), 3.64 (s, 3H), 3.23 (s, 3H), 2.15-2 , 24 (m, 1H), 1.56-1.76 (m, 2H), 1.31-1.55 (m, 11H), 1.02 (d, J = 6.6 Hz, 3H).
[0836] [0836] Step 2. Methyl syntheses N - {(2R, 3R) -3 - [(2S) -1- (tert-butoxycarbonyl) pyrrolidin-2-yl] -3-methoxy-2-methylpropanethiol} -L- phenylalaninate (# 38). A mixture of # 37 (1.52 g, 3.39 mmol, 1 eq.) And # 21 (1.68 g, 4.41 mmol, 1.3 eq.) In acetonitrile (12 mL, 0.28 M ) was subjected to microwave radiation at 100 ° C for 1 hour. The mixture was partitioned between water and ethyl acetate. The aqueous layer was re-extracted with ethyl acetate. The combined organic layers were washed with 10% aqueous citric acid solution and with brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The material was dissolved in a small amount of ethyl acetate and concentrated on silica in vacuo. Purification by chromatography on silica gel (Gradient: 0% to 30% ethyl acetate in heptane) provided # 38 (680 mg, 43%); LC-MS: m / z 465.2 [M + H +], 487.3 [M + Na +], 365.2 [(M - Boc) + H +], retention time = 0.97 minutes; HPLC (Protocol B): 465.2 [M + H +], 487.2 [M + Na +], 365.2 [(M - Boc) + H +], retention time = 7.444 minutes (purity> 98%); 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ 10.23 (br d, J = 7.5 Hz, 1H), 7.17-7.28 (m , 5H), 5.24 (ddd, J = 11, 7.5, 4.5 Hz, 1H), 3.66 (s, 3H), 3.28 (s, 3H), 3.21 (dd, J = 14.3, 4.4 Hz, 1H), 3.07 (dd, J = 14.2, 11.2 Hz, 1H), 2.65-2.74 (m, 1H), 1.54 -1.71 (m, 2H), 1.37 (s, 9H), 1.17 (d, J = 6.4 Hz, 3H).
[0837] [0837] Step 3. Synthesis of methyl-N - {(2R, 3R) -3-methoxy-2-methyl-3 - [(2S) -pyrrolidin-2-yl] propanothioyl} -L-phenylalaninate, hydrochloride salt (# 39). According to general procedure C, at 0 ° C from # 38 (660 mg, 1.42 mmol, 1 eq.), Dioxane (10 mL, 0.14 M) and 4 M of a hydrochloric acid solution in dioxane (20 mL, 80 mmol, 60 eq.) # 39 (590 mg) was synthesized as an off-white solid, which was used in the next step without further purification. LC-MS: m / z 365.2 [M + H +], retention time = 0.58 minutes; 1H NMR (400 MHz, DMSO-d6) δ 10.67 (d, J = 7.7 Hz, 1H), 9.42-9.54 (br m, 1H), 8.21-8.33 (br m, 1H), 7.20-7.35 (m, 5H), 5.25 (ddd, J = 11.1, 7.6, 4.4 Hz, 1H), 3.76 (dd, J = 8.9, 3.0 Hz, 1H), 3.68 (s, 3H), 3.39 (s, 3H), 3.24 (dd, J = 14.2, 4.5 Hz, 1H), 3.13 (dd, J = 14.3, 11.0 Hz, 1H), 2.93-3.09 (m, 3H), 2.85-2.93 (m, 1H), 1.72- 1.84 (m, 1H), 1.36-1.60 (m, 3H), 1.22 (d, J = 6.6 Hz, 3H).
[0838] [0838] Step 4. Synthesis of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -N-methyl-L-valyl-N- [(3R, 4S, 5S) -3-methoxy-1 - {( 2S) -2 - [(1R, 2R) -1-methoxy-3 - {[(2S) -1-methoxy-1-oxo-3-phenylpropan-2-yl] amino} -2-methyl-3-thioxopropyl ] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 40). According to general procedure D, from # 8 (247 mg, 0.387 mmol, 1 eq.), # 39 (186 mg, 0.450 mmol, 1.2 eq.), Dichloromethane (10 mL, 0.04 M ), N, N-dimethylformamide (2 mL), HATU (176 mg, 0.464 mmol, 1.2 eq) and triethylamine (189 µL, 1.35 mmol, 3.5 eq.), The desired crude material was synthesized, which was purified by silica gel chromatography (Gradient: 0% to 25% acetone in heptane) to provide # 40 (410 mg, 90% in 2 steps) as an off-white solid. LC-MS: m / z 984.7 [M + H +], 1006.7 [M + Na +], retention time = 1.15 minutes; HPLC (protocol C): retention time = 9.683 minutes (purity> 99%); 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ [10.19 (br d, J = 7 Hz) and 10.49 (br d, J = 8 Hz) , Total 1H], 7.90 (d, J = 7.5 Hz, 2H), 7.60-7.65 (m, 2H), 7.38-7.45 (m, 2H), 7.29 -7.35 (m, 2H), 7.14- 7.28 (m, 5H), [5.20 (ddd, J = 11, 7, 4 Hz) and 5.35-5.43 (m) , 1H total], 3.65 and 3.69 (2 s, 3H total), [1.15 (d, J = 6.5 Hz) and 1.18 (d, J = 6.4 Hz), 3H total].
[0839] [0839] Step 5A. Syntheses of N-methyl-L-valyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3- {[(1S) -1-carboxy-2 -phenylethyl] amino} -1-methoxy-2-methyl-3-thioxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide, trifluoroacetic acid salt
[0840] [0840] Step 5B. Syntheses of N-methyl-L-valyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2- [(1R, 2R) -1-methoxy-3 - {[( 2S) -1-methoxy-1-oxo-3-phenylpropan-2-yl] amino} -2-methyl-3-thioxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] - N-methyl-L-valinamide (# 42). According to general procedure A, from # 40 (561 mg, 0.570 mmol, 1 eq.), Dichloromethane (10 ml, 0.057 M) and diethylamine (10 ml), # 42 (348 mg, 80% ) as a white solid after chromatography on silica gel (Gradient: 0% to 10% methanol in dichloromethane). LC-MS: m / z 762.7 [M + H +], retention time = 0.74 minutes; HPLC (protocol A): 762.4 [M + H +], retention time = 7.315 minutes (purity> 95%); 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ [10.20 (br d, J = 7.5 Hz) and 10.50 (br d, J = 8 Hz),
[0841] [0841] Preparation of 2-Methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1- carboxy-2- phenylethyl] amino} -1-methoxy-2-methyl-3-thioxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide, salt hydrochloride (# 44) and 2-methylalanyl-N- [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-3 - {[ (2S) -1-methoxy-1-oxo-3-phenylpropan-2-yl] amino} -2-methyl-3-thioxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N- methyl-L-valinamide, hydrochloride salt (# 45)! *%, - (+! & (&. (
[0842] [0842] Step 1. Syntheses of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -2-methylalanyl-N- [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-3 - {[(2S) -1-methoxy-1-oxo-3-phenylpropan-2-yl] amino} -2-methyl-3-thioxopropyl] pyrrolidin-1 -il} -5-methyl-1-oxoheptan-4-yl] -N-
[0843] [0843] Step 2A. Syntheses of 2-methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3- {[(1S) -1-carboxy-2-phenylethyl] amino } -1-methoxy-2-methyl-3-thioxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide, hydrochloride salt ( # 44). To a solution of # 43 (100 mg, 0.105 mmol, 1 eq.) In tetrahydrofuran (5 mL, 0.02 M) was added a solution of lithium hydroxide (10 mg, 0.417 mmol, 3 eq.) In water ( 3 mL). After 3 hours, the reaction was concentrated in vacuo and purified by reverse phase chromatography (Method C) to provide a salt of trifluoroacetic acid, which was dissolved in methanol, treated with 4 M of a solution of hydrochloric acid in dioxane, and concentrated in vacuo to provide # 44 (56 mg, 71%) as a white solid. LC-MS: m / z 720.6 [M + H +], retention time = 0.67 minutes; HPLC (Protocol D): retention time = 8.851 minutes; 1H NMR (400 MHz, CD3OD), presumed to be a mixture of rotamers, characteristic signals: δ 7.17-7.31 (m, 5H), 3.34 and 3.35 (2 s, 3H total), 3.10 and 3.16 (2 br, 3H total), 1.62 and 1.64 (2 s, 3H total), 1.53 and 1.55 (2 s, 3H total), [1.26 (d, J = 6.5 Hz) and 1.30 (d, J = 6.5 Hz), total 3H], 0.84-0.91 (m, 3H).
[0844] [0844] Step 2B. Syntheses of 2-methylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2- [(1R, 2R) -1-methoxy-3 - {[(2S) -1 -methoxy-1-oxo-3-phenylpropan-2-yl] amino} -2-methyl-3-thioxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl- L-valinamide, hydrochloride salt (# 45). According to general procedure A, from # 43 (176 mg, 0.184 mmol, 1 eq.), Dichloromethane (4 mL, 0.05 M) and diethylamine (4 mL), the desired crude material was synthesized, which was purified by reverse phase chromatography (Method C). The resulting trifluoroacetic acid salt was dissolved in methanol, treated with a 4 M hydrochloric acid solution in dioxane, and concentrated in vacuo to provide # 45 (100 mg, 70%) as a white solid. LC-MS: m / z 734.6 [M + H +], retention time = 0.72 minutes; 1H NMR (400 MHz, CD3OD), presumed to be a mixture of rotamers, characteristic signals: δ 7.18-7.31 (m, 5H), 5.41 - 5.47 and 5.55-5.62 (2 m, 1H total), 3.73 and 3.76 (2 s, 3H total), 3.35 and 3.36 (2 s, 3H total), 3.10 and 3.15 (2 br s, 3H total), 1.62 and 1.64 (2 s, 3H total), 1.53 and 1.55 (2 s, 3H total), [1.25 (d, J = 6.6 Hz) and 1 , 29 (d, J = 6.5 Hz), total 3H], 0.84-0.91 (m, 3H).
[0845] [0845] Preparation of N2 - [(1-Aminocyclopentyl) carbonyl] -N - [(3R, 4S, 5S) -3-methoxy-1- {(2S) -2 - [(1R, 2R) -1-methoxy -2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} -5-methyl -1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 47)
[0846] [0846] Step 1. Synthesis of N2 - [(1 - {[(9H-fluoren-9-ylmethoxy) carbonyl] amino} cyclopentyl) -carbonyl] -N - [(3R, 4S, 5S) -3-methoxy- 1 - {(2S) -2- [(1R, 2R) -1-methoxy-2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2 - yl) ethyl] amino} propyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 46).
[0847] [0847] Step 2. Synthesis of N2 - [(1-aminocyclopentyl) carbonyl] -N - [(3R, 4S, 5S) -3- methoxy-1 - {(2S) -2 - [(1R, 2R) - 1-methoxy-2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} - 5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 47).
[0848] [0848] Preparation of N2 - [(1-Aminocyclopropyl) carbonyl] -N - [(3R, 4S, 5S) -3-methoxy-1- {(2S) -2 - [(1R, 2R) -1-methoxy -2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} -5-methyl -1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 51) and 1-amino-N - [(2S) -1 - {[(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2- il) ethyl] amino} propyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] (methyl) amino} -3-methyl-1-oxobutan-2-yl] cyclohexanecarboxamide (# 52)
[0849] [0849] Step 1. Synthesis of (2R, 3R) -3-methoxy-2-methyl-N - [(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] - 3 - [(2S) -pyrrolidin-2-yl] propanamide, trifluoroacetic acid salt and (3R, 4S, 5S) -4 - [{N - [(9H-fluoren-9-ylmethoxy) carbonyl] -L-valyl } (methyl) amino] -3-methoxy-5-methylheptanoic (# 48). To a solution of # 16 (1.0 g, 2.11 mmol, 1 eq.) And # 5 (1.22 g, 2.11 mmol, 1 eq.) In dichloromethane (20 mL, 0.1 M) at 0 ° C, trifluoroacetic acid (6 ml) was added. After 3 hours, the mixture was concentrated in vacuo to provide mixture # 48 (1.8 g), which was used in the next step without further purification; LC-MS (Protocol K): m / z 374.2 [M + H +], retention time = 2.093 minutes, 525.2 [M + H +], retention time = 4.875 minutes.
[0850] [0850] Step 2. Synthesis of N2 - [(9H-fluoren-9-ylmethoxy) carbonyl] -N - [(3R, 4S, 5S) -3- methoxy-1 - {(2S) -2 - [(1R , 2R) -1-methoxy-2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1 -yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 49).
[0851] [0851] Step 3. Syntheses of N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3-oxo -3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl ] -N-methyl-L-valinamide (# 50). In accordance with general procedure A, from # 49 (0.70 g, 0.79 mmol, 1 eq.), Dichloromethane (15 mL, 0.05 M) and diethylamine (10 mL) was synthesized # 50 (160 mg, 30%), after purification by chromatography on silica gel (Gradient: 0% to 5% methanol in dichloromethane). Rf 0.4 (10% methanol in dichloromethane); LC-MS (Protocol K): m / z 658.3 [M + H +], 680.3 [M + Na +], retention time = 2.760 minutes.
[0852] [0852] Step 4A. Syntheses of N2 - [(1-aminocyclopropyl) carbonyl] -N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2- methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} -5-methyl-1- oxoheptan-4-yl] -N-methyl-L-valinamide (# 51).
[0853] [0853] Step 4B. Syntheses of 1-amino-N - [(2S) -1 - {[(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2- [(1R, 2R) -1-methoxy-2 -methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} -5-methyl-1 -oxoheptan-4-yl] (methyl) amino} -3-methyl-1-oxobutan-2-yl] cyclohexanecarboxamide (# 52). To a solution of # 50 (120 mg, 0.18 mmol, 1 eq.), Brop (84 mg, 0.21 mmol, 1.2 eq.) And diisopropylethylamine (0.1 mL, 0.54 mmol, 3 eq.) in dichloromethane (15 ml, 0.009 M), at 0 ° C 1-aminocyclohexanecarboxylic acid (31 mg, 0.21 mmol, 1.2 eq.) was added. After 2 hours, the mixture was quenched with water and extracted twice with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by chromatography on silica gel (Gradient: 0% to 5% methanol in dichloromethane) to provide # 52 (50 mg, 35%).
[0854] [0854] Preparation of 2-Methylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1- methoxy-2-methyl-3- oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} - 5-methyl-1-oxoheptan-4- il] -N-methyl-L-valinamide (# 54)
[0855] [0855] Step 1. Syntheses of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -2-methylalanyl-N- [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl ] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 53). According to general procedure D, from # 32 (2.05 g, 2.83 mmol, 1 eq.) In dichloromethane (20 mL, 0.1 M) and N, N-dimethylformamide (3 mL), amine # 19 (2.5 g, 3.4 mmol, 1.2 eq.), HATU (1.29 g, 3.38 mmol, 1.2 eq.) and triethylamine (1.57 mL, 11, 3 mmol, 4 eq.), The desired crude material was synthesized, which was purified by chromatography on silica gel (Gradient: 0% to 55% acetone in heptane), producing # 53 (2.42 g, 74%) as a solid. LC-MS: m / z 965.7 [M + H +], 987.6 [M + Na +], retention time = 1.04 minutes; HPLC (protocol A): m / z 965.4 [M + H +], retention time = 11.344 minutes (purity> 97%); 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ 7.86-7.91 (m, 2H), [7.77 (d, J = 3.3 Hz ) and 7.79 (d, J = 3.2 Hz), total 1H], 7.67-7.74 (m, 2H), [7.63 (d, J = 3.2 Hz) and 7, 65 (d, J = 3.2 Hz), total 1H], 7.38-7.44 (m, 2H), 7.30- 7.36 (m, 2H), 7.11-7.30 ( m, 5H), [5.39 (ddd, J = 11.4, 8.4, 4.1 Hz) and 5.52 (ddd, J = 11.7, 8.8, 4.2 Hz), 1H total], [4.49 (dd, J = 8.6, 7.6 Hz) and 4.59 (dd, J = 8.6, 6.8 Hz), 1H total], 3.13, 3 , 17, 3.18 and 3.24 (4 s, 6H total), 2.90 and 3.00 (2 br s, 3H total), 1.31 and 1.36 (2 br s, 6H total), [1.05 (d, J = 6.7 Hz) and 1.09 (d, J = 6.7 Hz), total 3H].
[0856] [0856] Step 2. Synthesis of 2-methylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2- [(1R, 2R) -1-methoxy-2-methyl -3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan -4-yl] -N-methyl-L-valinamide (# 54).
[0857] [0857] Preparation of 2-Methylalanyl-N - {(3R, 4S, 5S) -3-methoxy-1 - [(2S) -2 - {(1R, 2R) -1- methoxy-2-methyl-3- oxo-3 - [(2-phenylethyl) amino] propyl} pyrrolidin-1-yl] -5-methyl-1-oxoheptan-4-yl} -N-methyl-L-valinamide, acetic acid salt (# 56)
[0858] [0858] Step 1. Syntheses of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -2-methylalanyl-N- {(3R, 4S, 5S) -3-methoxy-1 - [(2S) -2 - {(1R, 2R) -1-methoxy-2-methyl-3-oxo-3 - [(2-phenylethyl) amino] propyl} pyrrolidin-1-yl] -5-methyl-1-oxoheptan-4-yl } -N-methyl-L-valinamide (# 55). To a solution of # 24 (104 mg, 0.256 mmol, 1 eq.) In dichloromethane (10 ml, 0.094 M) was added # 32 (156 mg, 0.256 mmol, 0.9 eq.), Diisopropylethylamine (135 µL, 0.768 mmol, 3 eq.) and HATU (120 mg, 0.307 mmol, 1.2 eq.). After stirring for 18 hours, the mixture was concentrated in vacuo and the residue was diluted with ethyl acetate (10 ml), washed with 1 M aqueous hydrochloric acid solution (2 x ml) and with brine, dried over sodium sulfate , filtered, and concentrated in vacuo.
[0859] [0859] Step 2. Synthesis of 2-methylalanyl-N - {(3R, 4S, 5S) -3-methoxy-1 - [(2S) -2- {(1R, 2R) -1-methoxy-2-methyl -3-oxo-3 - [(2-phenylethyl) amino] propyl} pyrrolidin-1-yl] -5-methyl-1-oxoheptan-4-yl} -N-methyl-L-valinamide, acetic acid salt ( # 56). To a mixture of # 55 (44 mg, 0.050 mmol, 1 eq.) In tetrahydrofuran (1 ml, 0.05 M) was added diethylamine (0.5 ml). After stirring for 18 hours, the reaction mixture was concentrated in vacuo and the residue was purified by reverse phase chromatography (Method B) to provide # 56 (16.2 mg, 49%) as a solid. LC-MS: m / z 660.8 [M
[0860] [0860] Preparation of 2-Methylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1- methoxy-2-methyl-3- oxo-3 - {[((1-phenylcyclopropyl) methyl] amino} propyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 60) $% &&&&&&&&&&&&&&&&&&&&&&&&&&&
[0861] [0861] Step 1. Syntheses of 1- (1-phenylcyclopropyl) methanamine # @ 1. To a solution of 1-phenylcyclopropanecarbonitrile (50 g, 0.34 mol, 1 eq.) In tetrahydrofuran (500 mL, 0.7 M) at 0 ° C, was added lithium aluminum hydride (23 g, 0.35 mol) , 1.03 eq.). The reaction mixture was stirred at 0 ° C for one hour and then at reflux for one hour. The reaction mixture was then cooled and quenched with water (23 ml) and a 15% aqueous sodium hydroxide solution (69 ml). The mixture was filtered and concentrated in vacuo to provide # @ 1 (36 g, 72%). LC-MS: m / z 148.1 [M + H +], retention time = 0.86 minutes; 1H NMR (400 MHz, CDCl3) δ 7.2-7.4 (m, 5H), 2.78 (s, 2H), 1.19 (br s, 2H), 0.72-0.84 (m , 4H).
[0862] [0862] Step 2. Synthesis of tert-butyl (2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3-oxo-3- {[((1-phenylcyclopropyl) methyl] amino} propyl] pyrrolidine-1-carboxylate (# 57). According to general procedure D, from # 11 (2.15 g, 7.48 mmol, 1.1 eq.) In dichloromethane (20 mL, 0.3 M) and N, N-dimethylformamide (4 mL ), 1- (1-phenylcyclopropyl)
[0863] [0863] Step 3. Synthesis of (2R, 3R) -3-methoxy-2-methyl-N - [(1-phenylcyclopropyl) -methyl] - 3 - [(2S) -pyrrolidin-2-yl] propanamide, salt hydrochloride (# 58). According to general procedure C, from # 57 (566 mg, 1.36 mmol, 1 eq.) In dioxane (4 mL, 0.3 M) and 4 M of a solution of hydrochloric acid in dioxane (4 mL, 16 mmol, 11.7 eq.), # 58 (466 mg, 97%) was synthesized; LC-MS: m / z 318.2 [M + H +], 339.2 [M + Na +], retention time = 0.56 minutes; 1H NMR (400 MHz, DMSO-d6) δ 9.53 (br s, 1H), 8.48 (br s, 1H), 8.11 (br dd, J = 5.7, 5.6 Hz, 1H ), 7.23-7.30 (m, 4H), 7.14-7.21 (m, 1H), 3.58 (dd, J = 7.5, 3.9 Hz, 1H), 3, 50 (dd, J = 13.7, 6.3 Hz, 1H), 3.34 (s, 3H), 3.21 3.29 (br m, 1H), 3.18 (dd, J = 13 .8, 5.0 Hz, 1H), 3.04-3.13 (br m, 2H), 2.42-2.50 (m, 1H), 1.56-1.89 (m, 4H) , 1.04 (d, J = 6.9 Hz, 3H), 0.71-0.91 (m, 4H).
[0864] [0864] Step 4. Synthesis of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -2-methylalanyl-N- [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3-oxo-3 - {[(1-phenylcyclopropyl) methyl] amino} propyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan- 4-yl] -N-methyl-L-valinamide (# 59). According to general procedure D, from # 32 (550 mg, 0.902 mmol, 1 eq.), # 58 (350 mg, 0.992 mmol, 1.1 eq.), Dichloromethane (10 mL, 0.08 M ) and N, N-dimethylformamide (2 mL), HATU (446 mg, 1.17 mmol, 1.3 eq.) and triethylamine (0.503 mL, 3.61 mmol, 4 eq.), the desired crude material was synthesized , which was purified by silica gel chromatography (Gradient: 0% to 30% acetone in heptane), producing # 59 (618 mg, 69%) as an off-white solid. LC-MS: m / z 908.7 [M + H +], 930.7 [M + Na +], retention time = 1.07 minutes; HPLC (Protocol B at 45 ° C): m / z 908.5 [M + H +], retention time = 8.721 minutes (purity> 97%); 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ 7.89 (d, J = 7.5 Hz, 2H), 7.38-7.44 (m, 2H), 7.30- 7.36 (m, 2H), [4.49 (dd, J = 8.5, 7.8 Hz) and 4.59 (dd, J = 8.7, 6.9 Hz), 1H total], 4.18- 4.26 (m, 3H), 3.93-4.01 (br m, 1H), 3.23 and 3.26 (2 s, 3H total), 3 , 16 and 3.16 (2 s, 3H total), 2.91 and 3.05 (2 br s, 3H total), 1.36 and 1.37 (2 br s, 3H total), 1.30 and 1.32 (2 br s, 3H total), [1.00 (d, J = 6.7 Hz) and 1.02 (d, J = 6.6 Hz), 3H total], 0.67-0 , 78 (m, 7H).
[0865] [0865] Step 5. Synthesis of 2-methylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2- [(1R, 2R) -1-methoxy-2-methyl -3-oxo-3 - {[(1-phenylcyclopropyl) methyl] amino} propyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 60 ). According to general procedure A, from # 59 (605 mg, 0.666 mmol, 1 eq.), Dichloromethane (10 ml, 0.067 M) and diethylamine (10 ml), # 60 (379 mg, 83% ); HPLC (Protocol A at 45 ° C) m / z 685.5 [M + H +], retention time = 7.072 minutes; 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ [8.03 (br d, J = 9.6 Hz) and 8.07 (br d, J = 9 , 4 Hz), 1H total], [7.74 (br dd, J = 7, 4 Hz) and 7.99 (br dd, J = 5.9, 5.7 Hz), 1H total], 7, 20-7.27 (m, 4H), 7.11-7.17 (m, 1H), [4.49 (dd, J = 9, 7 Hz) and 4.58 (dd, J = 9, 7 , 5 Hz), 1H total], 3.96-4.04 (br m, 1H), 3.24 and 3.27 (2 s, 3H total), 3.18 and 3.19 (2 s, 3H total), 2.93 and 3.07 (2 br s, total 3H), 1.20 and 1.21 (2 s, total 3H), 1.12 and 1.14 (2 s, total 3H), [ 1.00 (d, J = 6.7 Hz) and 1.03 (d, J = 6.7 Hz), total 3H].
[0866] [0866] Preparation of 2-Methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[2- (cyclohepta-2,4,6 -trien-1-yl) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N- methyl-L-valinamide (# 66)
[0867] [0867] 4 M HCl in dioxane
[0868] [0868] Step 1. Syntheses of cyclohepta-2,4,6-trien-1-yl-acetonitrile (# 61). To a solution of anhydrous acetonitrile (3.12 mL, 56.2 mmol, 1 eq.) In tetrahydrofuran (281 mL, 0.2 M) was added lithium diisopropylamine (1.8 M in heptane / ethylbenzene / tetrahydrofuran, 31, 2 mL, 56.2 mmol, 1 eq.) At -78 ° C. After 20 minutes at -78 ° C, tropilium tetrafluoroborate (10 g, 56 mmol, 1 eq.) Was added. After minutes, the reaction was concentrated in vacuo and the residue was diluted with ethyl acetate and washed with water. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo to provide a brown oil, which was purified by chromatography on silica gel (Gradient: 0% to 10% ethyl acetate in heptane) to provide # 61 (1 , 88 g, 25%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 6.69-6.71 (m, 2H), 6.27-6.32 (m, 2H), 5.28-5.33 (m, 2H), 2, 61 (d, J = 7.2 Hz, 2H), 2.26 2.34 (m, 1H).
[0869] [0869] Step 2. Syntheses of 2- (cyclohepta-2,4,6-trien-1-yl) ethanamine (# 62). To a suspension of lithium aluminum hydride (911 mg, 24.0 mmol, 1.4 eq.) In anhydrous diethyl ether (75 mL, 0.23 M), at 0 ° C was added slowly, dropwise, over 15 minutes, a solution of # 61 (2.25 g, 17.2 mmol, 1 eq.) In diethyl ether (15 mL). The reaction was warmed to room temperature. After 5 hours, the reaction was cooled to 0 ° C and quenched by the addition of water (1 ml), then filtered through a small pad of Celite and washed with methanol. The filtrate was dried over sodium sulfate, filtered, and concentrated in vacuo to provide # 62 (1.683 g, 73%) as a golden oil. LC-MS: m / z 136.1 [M + H +], retention time = 0.23 minutes; 1H NMR (400 MHz, CDCl3) δ 6.64-6.67 (m, 2H), 6.16-6.21 (m, 2H), 5.16-5.21 (m, 2H), 2, 84-2.89 (m, 2H), 1.86-1.92 (m, 2H), 1.62-1.70 (m, 1H).
[0870] [0870] Step 3. Synthesis of tert-butyl (2S) -2 - [(1R, 2R) -3 - {[2- (cyclohepta-2,4,6-trien-1-yl) ethyl] amino} - 1-methoxy-2-methyl-3-oxopropyl] pyrrolidine-1-carboxylate (# 63). To a solution of # 11 (3.57 g, 12.4 mmol, 1 eq.) In dichloromethane (100 ml, 0.1 M) and N, N-dimethylformamide (4 ml) was added HATU (5.36 g , 13.7 mmol, 1.1 eq.). After 20 minutes, triethylamine (5.20 mL, 37.3 mmol, 3 eq.) Was added, followed by # 62 (1.68 g, 12.4 mmol, 1 eq.), And the mixture was stirred for 18 minutes. hours. The reaction was concentrated in vacuo and the residue was taken up in ethyl acetate and washed with water (50 ml). The aqueous layer was re-extracted with ethyl acetate (3 times) and the combined organic layers were dried, filtered, and concentrated in vacuo to provide a brown oil, which was purified by chromatography on silica gel (Gradient: 0% to 100% ethyl acetate in heptane) to provide # 63 (2.95 g, 59% yield) as a viscous oil. LC-MS: m / z 405.4 [M + H +], 427.4 [M + Na +], retention time = 0.75 minutes; 1H NMR (400 MHz, CDCl3), presumed to be a mixture of rotamers: δ 6.63-6.68 (m, 2H), 6.16-6.23 (m, 2H), 5.19 (br dd, J = 9.0, 5.8 Hz, 2H), 3.51-3.63 and 3.71-3.90 (2 br multiplets, 3H total), 3.42 (s, 3H), 3 , 18-3.29 and 3.34-3.47 (2 br multiplets, total 3H), 2.27-2.45 (br m, 1H), 1.6-2.00 (m, 7H), 1.47 and 1.50 (2 br s, total 9H), 1.16-1.29 (br m, 3H).
[0871] [0871] Step 4. Synthesis of (2R, 3R) -N- [2- (cyclohepta-2,4,6-trien-1-yl) ethyl] -3-methoxy-2-methyl-3 - [(2S ) -pyrrolidin-2-yl] propanamide, hydrochloride salt (# 64)
[0872] [0872] Intermediate # 63 (400 mg, 0.989 mmol, 1 eq.) Was treated with 4 M of a solution of hydrochloric acid in dioxane (10 mL, 40 mmol, 40 eq.). After 1 hour, the reaction mixture was concentrated in vacuo and the residue was taken up in dichloromethane and washed with 1 M sodium hydroxide solution. The aqueous layer was re-extracted with dichloromethane and the combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo to provide # 64 (301 mg, quantitative) as a brown oil, which slowly started to solidify on standing. LC-MS: m / z 305.3 [M + H +], retention time = 0.54 minutes; HPLC (Protocol G): retention time = 4.848 minutes; 1H NMR (400 MHz, CDCl3), characteristic signals: δ 6.64-6.67 (m, 2H), 6.16-6.22 (m, 2H), 6.08-6.14 (br m, 1H), 5.16-5.22 (m, 2H), 3.44 (s, 3H), 3.31 (dd, J = 6.3, 4.5 Hz, 1H), 2.98-3 , 04 (m, 1H), 2.94 (ddd, J = 10.5, 7.2, 5.6 Hz, 1H), 2.81 (ddd, J = 10.5, 7.7, 6, 7 Hz, 1H), 2.57 (qd, J = 7.1, 4.5 Hz, 1H), 1.90-1.97 (m, 2H), 1.49-1.55 (m, 1H ), 1.18 (d, J = 7.1 Hz, 3H).
[0873] [0873] Step 5. Syntheses of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -2-methylalanyl, N- [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R , 2R) -3 - {[2- (cyclohepta-2,4,6-trien-1-yl) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3 -methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 65). According to general procedure D, from # 32 (678 mg, 0.937 mmol, 1 eq.) In dichloromethane (9.37 mL, 0.1 M), amine # 64 (300 mg, 0.985 mmol, 1 , 1 eq.), HATU (427 mg, 1.12 mmol, 1.2 eq.) And diisopropylethylamine (494 µL, 2.81 mmol, 3 eq.), The desired crude material was synthesized, which was purified by chromatography on silica gel (Gradient: 0% to 50% acetone in heptane), producing # 65 (546 mg, 65%) as a solid. LC-MS: m / z 896.7 [M + H +], 918.7 [M + Na +], retention time = 1.06 minutes.
[0874] [0874] Step 6. Synthesis of 2-methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[2- (cyclohepta-2, 4,6-trien-1-yl) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 66). To a solution of # 65 (540 mg, 0.603 mmol, 1 eq.) In dichloromethane (10 ml, 0.06 M) was added triethylamine (10 ml) and the reaction mixture was stirred for 2 hours. The mixture was concentrated in vacuo and the residue was purified by chromatography on silica gel
[0875] [0875] Preparation of 2-Methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1- carboxy-2- phenylethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 69) and 2-methylalanyl-N - [(3R, 4S, 5S) -3-methoxy- 1 - {(2S) -2 - [(1R, 2R) -1-methoxy-3 - {[(2S) - 1-methoxy-1-oxo-3-phenylpropan-2-yl] amino} -2-methyl-3-oxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl -L-valinamide (# 70) $%
[0876] [0876] Step 1. Synthesis of methyl-N - {(2R, 3R) -3-methoxy-2-methyl-3 - [(2S) -pyrrolidin-2-yl] propanoyl} -L-phenylalaninate, hydrochloride salt (# 67). According to general procedure C, from # 37 (2.39 g, 5.33 mmol, 1 eq.), Dioxane (10 mL, 0.53 M) and 4 M of a solution of hydrochloric acid in dioxane (10 mL, 40 mmol, 7.5 eq.), # 67 (2.21 g) was synthesized as a white solid, which was used in the next step without further purification. LC-MS: m / z 349.2 [M + H +], retention time = 0.53 minutes; 1H NMR (400 MHz, DMSO-d6) δ 9.45-9.58 (br m, 1H), 8.63 (d, J = 8.1 Hz, 1H), 8.51-8.62 (br m, 1H), 7.25-7.33 (m, 4H), 7.18-7.25 (m, 1H), 4.50 (ddd, J = 10.8, 8.1, 4.5 Hz, 1H), 3.65 (s, 3H), 3.54 (dd, J = 6.8, 4.5 Hz, 1H), 3.20 (s, 3H), 3.11 (dd, J = 13.8, 4.5 Hz, 1H), 2.99-3.14 (br m, 3H), 2.89 (dd, J = 13.8, 10.9 Hz, 1H), 2.44 -2.50 (m, 1H, assumed; partially obscured by the solvent peak), 1.77-1.89 (m, 1H), 1.60-1.73 (m, 2H), 1.46-1 , 57 (m, 1H), 1.05 (d, J = 6.8 Hz, 3H).
[0877] [0877] Step 2. Syntheses of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -2-methylalanyl-N- [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-3 - {[(2S) -1-methoxy-1-oxo-3-phenylpropan-2-yl] amino} -2-methyl-3-oxopropyl] pyrrolidin-1 -yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 68). According to general procedure D, from # 32 (353 mg, 0.488 mmol, 1 eq.) In dichloromethane (10 mL, 0.04 M), amine # 67 (271 mg, 0.588 mmol, 1.3 eq. .), HATU (223 mg, 0.586 mmol, 1.2 eq.) And diisopropylethylamine (238 µL, 1.71 mmol, 3.5 eq.), The desired crude material was synthesized, which was purified by chromatography on silica gel (Gradient: 0% to 40% acetone in heptane), providing # 68 (404 mg, 88% over two steps) as a solid.
[0878] [0878] Step 3A. Syntheses of 2-methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3- {[(1S) -1-carboxy-2-phenylethyl] amino } -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide, trifluoroacetic acid salt (# 69). To a solution of # 68 (143 mg, 0.152 mmol, 1 eq.) In tetrahydrofuran (5 mL, 0.02 M) was added a solution of lithium hydroxide (9.10 mg, 0.378 mmol, 2.5 eq. ) in water (3 mL). After 5 hours, the reaction was concentrated in vacuo, subjected to azeotropic distillation three times with heptane, dissolved in dimethyl sulfoxide (2.2 mL) and purified by reverse phase chromatography (Method C) to provide # 69 (56 mg, 52 %). HPLC (Protocol A at 45 ° C): 704.4 [M + H +], retention time = 6.623 minutes; 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ 8.08-8.22 and 8.37-8.49 (2 m, 5H total), 7.12 -7.28 (m, 5H), 3.18, 3.20 and 3.24 (3 s, 6H total), 2.95 and 3.04 (2 br s, 3H total), 1.52 and 1 , 53 (2 s, 3H total), 1.39 and 1.41 (2 s, 3H total), [1.02 (d, J = 6.8 Hz) and 1.05 (d, J = 6, 6 Hz), total 3H], 0.74-0.81 (m, 3H).
[0879] [0879] Step 3B. Syntheses of 2-methylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2- [(1R, 2R) -1-methoxy-3 - {[(2S) -1 -methoxy-1-oxo-3-phenylpropan-2-yl] amino} -2-methyl-3-oxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl- L-valinamide (# 70). According to general procedure A, from # 68 (240 mg, 0.255 mmol, 1 eq.), Dichloromethane (10 ml, 0.026 M) and diethylamine (10 ml), # 70 (120 mg, 65% ) as a white solid / glass mixture after chromatography on silica gel (Gradient: 0% to 10% methanol in dichloromethane). HPLC (Protocol A at 45 ° C): m / z 762.7 [M + H +], 740.4 [M + Na +], retention time = 6.903 minutes; 1H NMR (400 MHz, DMSO-d6), is presumed to be a mixture of rotamers, characteristic signals: δ [8.26 (d, J = 8.1 Hz) and 8.49 (d, J = 8.3 Hz), total 1H], [8.03 (d, J =
[0880] [0880] Preparation of N2 - [(3-Aminooxetan-3-yl) carbonyl] -N - {(3R, 4S, 5S) -3-methoxy-1- [(2S) -2 - {(1R, 2R) -1-methoxy-2-methyl-3-oxo-3 - [(2-phenylethyl) amino] propyl} pyrrolidin-1-yl] -5-methyl-1-oxoheptan-4-yl} -N-methyl-L -valinamide, acetic acid salt (# 75) $% &&&&&&&&&&&&&&&& * & 8! # two . /
[0881] [0881] Step 1. Synthesis of 3 - [(tert-butoxycarbonyl) amino] oxetane-3-carboxylic acid (# 71). For 1-aminooxetan-3-carboxylic acid (1.00 g, 8.54 mmol, 1 eq.) In dioxane (15 mL, 0.5 M), a sodium hydroxide solution (1.55 g, 38 , 7 mmol, 4.5 eq.) In water (15 ml) followed by di-tert-butyl dicarbonate (2.09 g, 9.29 mmol, 1.1 eq.). A white solid formed. The reaction was stirred for 18 hours and then concentrated in vacuo. The residue was taken up in ethyl acetate and washed with 1 M aqueous hydrochloric acid solution and with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo to provide # 71 (633 mg, 38%) as a white solid. 1H NMR (400 MHz,
[0882] [0882] Step 2. Synthesis of N2 - [(9H-fluoren-9-ylmethoxy) carbonyl] -N - {(3R, 4S, 5S) -3- methoxy-1 - [(2S) -2 - {(1R , 2R) -1-methoxy-2-methyl-3-oxo-3 - [(2-phenylethyl) amino] propyl} pyrrolidin- 1-yl] -5-methyl-1-oxoheptan-4-yl} -N- methyl-L-valinamide (# 72). For # @ 5 (9.47 g, 18.0 mmol, 1 eq.) And # 24 (5.90 g, 18.0 mmol, 1 eq.) In dichloromethane (250 mL, 0.072 M), diisopropylethylamine ( 9.52 ml, 54.2 mmol, 3 eq.) And HATU (8.49 g, 21.7 mmol, 1.2 eq.). The reaction was stirred for 18 hours and then concentrated in vacuo. The residue was taken up in ethyl acetate (300 ml) and washed with 1 M aqueous hydrochloric acid solution (2 x 100 ml) and with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was taken up in dichloromethane (250 ml) and filtered. The filtrate was concentrated in vacuo on silica and purified by silica gel chromatography (Gradient: 0% to 50% acetone in heptane) to provide # 72 (11.61 g, 81%) as a light yellow solid. LC-MS: m / z 797.6 [M + H +], 819.6 [M + Na +], retention time = 1.06 minutes; 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ 3.26 and 3.28 (2 s, 3H total), 3.18 and 3.20 (2 s, Total 3H), 2.95 and 3.10 (2 br s, total 3H), 1.01-1.09 (m, 3H), 0.67-0.78 (m, 3H).
[0883] [0883] Step 3. Syntheses of N - {(3R, 4S, 5S) -3-methoxy-1 - [(2S) -2 - {(1R, 2R) -1-methoxy-2-methyl-3-oxo -3 - [(2-phenylethyl) amino] propyl} pyrrolidin-1-yl] -5-methyl-1-oxoheptan-4-yl} -N-methyl- L-valinamide (# 73). For # 72 (5.16 g, 6.47 mmol, 1 eq.) In tetrahydrofuran (10 mL, 0.65 M), diethylamine (10 mL) was added. After 2 hours, the reaction was concentrated in vacuo and the residue was purified by chromatography on silica gel (Gradient: 0% to 10% methanol in dichloromethane) to provide # 73 (2.414 mg, 65%). LC-MS: m / z 576.5 [M + H +], retention time = 0.64 minutes; 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signs: δ 7.80-7.88 and 7.99-
[0884] [0884] Step 4. Synthesis of N2 - ({3 - [(tert-butoxycarbonyl) amino] oxetan-3-yl} carbonyl) - N - {(3R, 4S, 5S) -3-methoxy-1 - [( 2S) -2 - {(1R, 2R) -1-methoxy-2-methyl-3-oxo-3 - [(2-phenylethyl) amino] propyl} pyrrolidin-1-yl] -5-methyl-1-oxoheptan -4-yl} -N-methyl-L-valinamide (# 74). For # 73 (100 mg, 0.174 mmol, 1 eq.) In dichloromethane (4 mL, 0.04) and N, N-dimethylformamide (0.5 mL), # 71 (45.2 mg, 0.208 mmol, 1.2 eq.), Followed by diisopropylethylamine (92 µL, 0.521 mmol, 3 eq.) And HATU (102 mg, 0.260 mmol, 1.5 eq.). After 16 hours, the reaction was concentrated in vacuo and the residue was taken up in ethyl acetate (6 ml) and washed with 1 M aqueous hydrochloric acid solution (2 x 2 ml) and with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified by reverse phase chromatography (Method C) to provide # 74 (140 mg), which was used in the next step without further purification. LC-MS: m / z 774.7 [M + H +], 796.6 [M + Na +], retention time = 0.91 minutes.
[0885] [0885] Step 5. Synthesis of N2 - [(3-aminooxetan-3-yl) carbonyl] -N - {(3R, 4S, 5S) -3-methoxy-1 - [(2S) -2 - {(1R , 2R) -1-methoxy-2-methyl-3-oxo-3 - [(2-phenylethyl) amino] propyl} pyrrolidin- 1-yl] -5-methyl-1-oxoheptan-4-yl} -N- methyl-L-valinamide, acetic acid salt (# 75). For # 74 (140 mg, 0.181 mmol, 1 eq.) In dichloromethane (3 ml, 0.06 M), trifluoroacetic acid (1 ml) was added. After 1 hour, the reaction was concentrated in vacuo and the residue was taken up in ethyl acetate (6 ml) and washed with saturated aqueous sodium bicarbonate solution (2 ml) and with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. Half of the crude material was purified by reverse phase chromatography (Method B) to provide # 75 (16 mg, 26%, over two steps). LC-MS: m / z 674.6 [M + H +], retention time = 0.68 minutes; HPLC (Protocol A at 45 ° C): m / z 674.5 [M + H +], retention time = 7.128 minutes; 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signs: δ 7.80-7.87 and 8.02-8.07 (2 m, 2H total), 7.23 -7.30 (m, 2H), 7.14-7.22 (m, 3H), 4.28-4.33 (m, 2H), 3.96-4.04 (br m, 1H), 3.17 and 3.19 (2 s, 3H total), 2.96 and 3.10 (2 br s, 3H total), [1.04 (d, J = 7.0 Hz) and 1.07 ( d, J = 6.6 Hz), total 3H].
[0886] [0886] Preparation of N, 2-Dimethylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) - 1-methoxy-3 - {[ (2S) -1-methoxy-1-oxo-3-phenylpropan-2-yl] amino} -2-methyl-3-thioxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 79) and N, 2-dimethylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[ (1S) -1-carboxy-2-phenylethyl] amino} -1- methoxy-2-methyl-3-thioxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl- L-valinamide, trifluoroacetic acid salt (# 80)
[0887] [0887] Step 1. Methyl-N syntheses - {(2R, 3R) -3 - [(2S) -1- {(3R, 4S, 5S) -4 - [{N - [(9H- fluoren-9 -ylmethoxy) carbonyl] -L-valyl} (methyl) amino] -3-methoxy-5-methylheptanoyl} pyrrolidin-2-yl] -3-methoxy-2-methylpropanothioyl} -L-phenylalaninate (# 76). According to general procedure D, from # @ 5 (260 mg, 0.648 mmol, 1 eq.), # 39 (340 mg, 0.629 mmol, 1 eq.), Dichloromethane (10 mL, 0.065 M), HATU (296 mg, 0.778 mmol, 1.2 eq.) And diisopropylethylamine (339 µL, 1.94 mmol, 3 eq.), The desired crude material was synthesized, which was purified by silica gel chromatography (Gradient: 0% to 40% acetone in heptane) to provide # 76 (466 mg, 83% over two steps) as a solid. LC-MS: m / z 871.5 [M + H +], 893.5 [M + Na +], retention time = 1.10 minutes; HPLC (protocol C): retention time = 9.249 minutes (purity> 99%); 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ [10.19 (br d, J = 7.4 Hz) and 10.49 (br d, J = 7 , 8 Hz), total 1H], 7.89 (br d, J = 7.4 Hz, 2H), 7.68-7.75 (m, 2H), 7.54-7.60 (m, 1H ), 7.41 (br dd, J = 7.4, 7.4 Hz, 2H), 7.28-7.36 (m, 2H), 7.15-7.28 (m, 5H), [ 5.20 (ddd, J = 10.9, 7.3, 4.4 Hz) and 5.34-5.43 (m), 1H total], 3.65 and 3.69 (2 s, 3H total ), 3.24 and 3.25 (2 s, 3H total), 3.17 (br s, 3H), 2.93 and 2.98 (2 br s, 3H total), [1.15 (d, J = 6.6 Hz) and 1.18 (d, J = 6.6 Hz), total 3H].
[0888] [0888] Step 2. Methyl syntheses N - {(2R, 3R) -3-methoxy-3 - [(2S) -1 - {(3R, 4S, 5S) -3- methoxy-5-methyl-4- [methyl (L-valyl) amino] heptanoyl} pyrrolidin-2-yl] -2-methylpropanothioyl} -L-phenylalaninate (# 77). According to general procedure A, from # 76 (460 mg, 0.528 mmol, 1 eq.) Tetrahydrofuran (8 ml, 0.07 M) and diethylamine (8 ml), # 77 (399 mg) was synthesized, which was used in the next step without further purification; LC-MS m / z 649.5 [M + H +], retention time = 0.73 minutes; 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic product signals: δ [10.20 (d, J = 7.4 Hz) and 10.49 (d, J = 7 , 4 Hz), 1H total], 7.15 -7.28 (m, 5H), [5.20 (ddd, J = 10.9, 7.2, 4.5 Hz) and 5.34-5 , 42 (m), 1H total], 3.65 and 3.68 (2 s, 3H total), 3.24 and 3.25 (2 s, 3H total), 3.15 and 3.15 (2 s , 3H total), 2.83 and 2.88 (2 br s, 3H total), [1.15 (d, J = 6.6 Hz) and 1.18 (d, J = 6.6 Hz), Total 3H].
[0889] [0889] Step 3. Synthesis of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -N, 2-dimethylalanyl-N- [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-3 - {[(2S) -1-methoxy-1-oxo-3-phenylpropan-2-yl] amino} -2-methyl-3-thioxopropyl] pyrrolidin -1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 78). According to general procedure D, from # 77 (399 mg, 0.52 mmol, 1 eq.), N - [(9H-fluoren-9-ylmethoxy) carbonyl] -N, 2-dimethylalanine (213 mg , 0.628 mmol, 1.2 eq.), Dichloromethane (5 mL, 0.1 M), HATU (239 mg, 0.628 mmol, 1.2 eq.) And diisopropylethylamine (282 µL, 1.62 mmol, 3.1 eq.), the desired crude material was synthesized, which was purified by silica gel chromatography (Gradient: 0% to 50% acetone in heptane), providing # 78 (231 mg, 46% over two steps). LC-MS: m / z 970.7 [M + H +], 992.6 [M + Na +], retention time = 1.11 minutes; HPLC (Protocol C): retention time = 9.260 minutes; 1 H NMR (400 MHz, DMSO-d6), is presumed to be a mixture of rotamers, characteristic signals: δ [10.19 (d, J = 7.4 Hz) and 10.47 (d, J = 7, 8 Hz), 1H total], 7.89 (d, J = 7.4 Hz, 2H), 7.61-7.67 (m, 2H), 7.41 (br dd, J = 7.4, 7.4 Hz, 2H), 7.14-7.36 (m, 8H), [5.20 (ddd, J = 11, 7, 5 Hz) and 5.38 (ddd, J = 11, 8, 4 Hz), 1H total], [4.41 (dd, J = 8.6, 8.4 Hz) and 4.46 (dd, J = 8.2, 8.2 Hz), 1H total], 3 , 65 and 3.68 (2 s, 3H total), 3.23 and 3.24 (2 s, 3H total), 3.13 (br s, 3H), 2.88 and 2.93 (2 br s , 3H total), 2.84 and 2.85 (2 s, 3H total), 1.31 and 1.32 (2 s, 3H total), [1.15 (d, J = 6.6 Hz) and 1.18 (d, J = 6.4 Hz), total 3H].
[0890] [0890] Step 4A. Syntheses of N, 2-dimethylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2- [(1R, 2R) -1-methoxy-3 - {[(2S) -1-methoxy-1-oxo-3-phenylpropan-2-yl] amino} -2-methyl-3-thioxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N- methyl-L-valinamide (# 79). In accordance with general procedure A, from # 78 (223 mg, 0.230 mmol, 1 eq.), Dichloromethane (6 ml, 0.04 M) and diethylamine (6 ml), # 79 (146 mg, 85%) as a white solid after chromatography on silica gel (Gradient: 0% to 5% methanol in heptane, then 0% to 10% methanol in dichloromethane). HPLC (Protocol A at 45 ° C): 749.4 [M + H +], retention time = 7.315 minutes; 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ [10.20 (d, J = 7.6 Hz) and 10.50 (d, J = 8.0 Hz), 1H total], 7.79- 7.88 (m, 1H), 7.15-7.29 (m, 5H), [5.20 (ddd, J = 11, 7 Hz) and 5.38 (ddd, J = 11, 8, 4 Hz), total 1H], [4.50 (dd, J = 8.8, 8.6 Hz) and 4.56 (dd, J = 9, 8 Hz), 1H total], 3.65 and 3.69 (2 s, 3H total), 3.24 and 3.25 (2 s, 3H total), 3.16 (br s, 3H), 2.93 and 2.97 (2 br, 3H total), 2.10 and 2.11 (2 s, 3H total).
[0891] [0891] Step 4B. Syntheses of N, 2-dimethylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3- {[(1S) -1-carboxy-2-phenylethill ] amino} -1-methoxy-2-methyl-3-thioxopropyl] pyrrolidin-1-yl} -3-
[0892] [0892] Preparation of N, 2-Dimethylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) - 1-methoxy-2-methyl- 3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} -3-thioxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4 -il] -N-methyl-L-valinamide (# 84)! /
[0893] [0893] Step 1. Synthesis of N2 - [(9H-fluoren-9-ylmethoxy) carbonyl] -N - [(3R, 4S, 5S) -3- methoxy-1 - {(2S) -2 - [(1R , 2R) -1-methoxy-2-methyl-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} -3-thioxopropyl] pyrrolidin-1- il} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 81). According to general procedure D, from # @ 5 (620 mg, 1.18 mmol, 1 eq.), Dichloromethane (10 mL, 0.1 M), amine # 18 (604 mg, 1.42 mmol , 1.2 eq.), Diisopropylethylamine (618 µL, 3.54 mmol, 3 eq.) And HATU (539 mg, 1.42 mmol, 1.2 eq.), The desired crude material was synthesized, which was purified chromatography on silica gel (Gradient: 0% to 30% acetone in heptane) to provide # 81 (737 mg, 58%). HPLC (Protocol C): retention time = 9.235 minutes; 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ [10.54 (br d, J = 8 Hz) and 10.81 (br d, J = 8 Hz) , Total 1H], 7.89 (d, J = 7.6 Hz, 2H), [7.80 (d, J = 3.3 Hz) and 7.83 (d, J = 3.1 Hz), Total 1H], 7.70-7.75 (m, 2H), [7.64 (d, J = 3.1 Hz) and 7.68 (d, J = 3.3 Hz), total 1H], 7.55-7.60 (m, 1H), 7.38-7.44 (m, 2H), 7.13- 7.35 (m, 7H), [6.31 (ddd, J = 11, 8, 4.5 Hz) and 6.40-6.48 (m), 1H total], 3.23 and 3.24 (2 s, 3H total), 3.17 and 3.22 (2 s, 3H total), 2.94 and 3.01 (2 br s, 3H total), [1.14 (d, J = 6.4 Hz) and 1.17 (d, J = 6.2 Hz), total 3H ].
[0894] [0894] Step 2. Synthesis of N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3- {{ [(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} -3-thioxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 82). According to general procedure A, from # 81 (733 mg, 0.818 mmol, 1 eq.) In dichloromethane (7 mL, 0.1 M) and diethylamine (7 mL), # 82 (670 mg) was synthesized , which was used in the next step without further purification. LC-MS: m / z 674.5 [M + H +], retention time = 1.29 minutes; 1H NMR (400 MHz, DMSO-d6), presumably a mixture of rotamers, characteristic product signals: δ [10.55 (br d, J = 8 Hz) and 10.84 (br d, J = 8 Hz), 1H total], [7.64 (d, J = 3.1 Hz) and 7.69 (d, J = 3.3 Hz), 1H total], 7.13-7.33 (m, 5H), 6.27-6.35 and 6.38-6.47 (2 m, 1H total), 3.23 and 3.25 (2 s, 3H total), 3.15 and 3.19 (2 s, 3H total), 2.84 and 2.91 (2 br s, 3H total).
[0895] [0895] Step 3. Syntheses of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -N, 2-dimethylalanyl-N- [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} -3- thioxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 83). According to general procedure D, from # 82 (670 mg, <0.818 mmol, 1 eq.), Dichloromethane (5 mL, 0.16 M), N - [(9H-fluoren-9-ylmethoxy) carbonyl ] -N, 2-dimethylalanine (304 mg, 0.896 mmol, 1.1 eq.), HATU (372 mg, 0.978 mmol, 1.2 eq.) And diisopropylethylamine (440 µL, 2.53 mmol, 3.1 eq. .), the desired crude material was synthesized, which was purified by silica gel chromatography (Gradient: 0% to 30% acetone in heptane) to provide # 83 (556 mg, 69% over two steps). LC-MS: m / z 994.7 [M + H +], retention time = 0.69 minutes; HPLC (protocol C): retention time = 9.333 minutes (purity> 98%); 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ [10.53 (br d, J = 8 Hz) and 10.80 (br d, J = 8 Hz) , Total 1H], 7.86-7.91 (m, 2H), [7.80 (d, J = 3.3 Hz) and 7.82 (d, J = 3.2 Hz), total 1H] , [7.64 (d, J = 3.2 Hz) and 7.68 (d, J = 3.2 Hz), total 1H], 7.62-7.66 (m, 2H), 7.38 -7.44 (m, 2H), 7.28- 7.36 (m, 5H), 7.19-7.26 (m, 2H), 7.12-7.17 (m, 1H), [ 6.31 (ddd, J = 11, 8, 4.5 Hz) and 6.44 (ddd, J = 11, 8.5, 4.5 Hz), total 1H], [4.42 (dd, J = 9, 8 Hz) and 4.48 (dd, J = 8, 8 Hz), 1H total], 3.22 and 3.24 (2 s, 3H total), 3.13 and 3.17 (2 s , 3H total), 2.89 and 2.97 (2 br s, 3H total), 2.84 and 2.85 (2 s, 3H total), [1.13 (d, J = 6.4 Hz) and 1.16 (d, J = 6.4 Hz), total 3H].
[0896] [0896] Step 4. Synthesis of N, 2-dimethylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2- [(1R, 2R) -1-methoxy-2 -methyl-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} -3-thioxopropyl] pyrrolidin-1-yl} -5-methyl-1- oxoheptan-4-yl] -N-methyl-L-valinamide (# 84). According to general procedure A, from # 83 (552 mg, 0.555 mmol, 1 eq.) In dichloromethane (10 mL, 0.05 M) and diethylamine (10 mL), the desired crude material, the which was diluted with methanol, concentrated in vacuo on silica, and purified by silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) to provide # 84 (406 mg, 95%) as a white solid. LC-MS: m / z 772.8 [M + H +], retention time = 1.35 minutes; HPLC (protocol A): 774.4 [M + H +], retention time = 7.390 minutes; 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ [10.54 (br d, J = 8 Hz) and 10.81 (br d, J = 8 Hz) , 1H total], 7.78-7.84 (m, 2H), [7.65 (d, J = 3.1 Hz) and 7.69 (d, J = 3.3 Hz), total 1H] , 7.29-7.34 (m, 2H), 7.20-7.28 (m, 2H), 7.14-7.19 (m, 1H), 6.27-6.35 and 6, 40-6.48 (2 m, 1H total), [4.51 (dd, J = 9, 8 Hz) and 4.57 (dd, J = 9, 8 Hz), 1H total], 3.24 and 3.25 (2 s, 3H total), 3.16 and 3.21 (2 s, 3H total), 2.94 and 3.00 (2 br s, 3H total), 2.09 and 2.10 ( 2 s, 3H total), 1.08 and 1.09 (2 s, 3H total), 0.73-0.80 (m, 3H).
[0897] [0897] N, 2-Dimethylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3- oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4- il] -N-methyl-L-valinamide (# 88) 9,%:
[0898] [0898] Step 1. Synthesis of N2 - [(9H-fluoren-9-ylmethoxy) carbonyl] -N - [(3R, 4S, 5S) -3- methoxy-1 - {(2S) -2 - [(1R , 2R) -1-methoxy-2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1 -yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 85).
[0899] [0899] Step 2. Syntheses of N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3-oxo -3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl ] -N-methyl-L-valinamide (# 86). According to general procedure A, from # 85 (5.00 g, 5.68 mmol, 1 eq.) In tetrahydrofuran (10 mL, 0.56 M) and diethylamine (3 mL), the material was synthesized desired crude, which was purified by silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) to provide # 86 (2.952 g, 79%) as a solid. LC-MS: m / z 658.5 [M + H +], 680.5 [M + Na +] retention time = 0.66 minutes; 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ [8.64 (d, J = 8.4 Hz) and 8.90 (br d, J = 8, 8 Hz), 1H total], [7.77 (d, J = 3.3 Hz) and 7.80 (d, J = 3.3 Hz), 1H total], [7.63 (d, J = 3.3 Hz) and 7.66 (d, J = 3.3 Hz), total 1H], 7.12-7.31 (m, 5H), [5.39 (ddd, J = 11.2, 8.4, 4.2 Hz) and 5.54 (ddd, J = 11.9, 8.9, 4.0 Hz), total 1H], 3.15, 3.19, 3.20 and 3, 26 (4 s, 6H total), 2.86 and 2.98 (2 br s, 3H total), [1.06 (d, J = 6.6 Hz) and 1.11 (d, J = 6, 6 Hz), 3H total].
[0900] [0900] Step 3. Syntheses of N- (tert-butoxycarbonyl) -N, 2-dimethylalanyl-N - [(3R, 4S, 5S) - 3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1- il} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 87).
[0901] [0901] Step 4. Synthesis of N, 2-dimethylalanyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2- [(1R, 2R) -1-methoxy-2 -methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} -5-methyl-1 -oxoheptan-4-yl] -N-methyl-L-valinamide (# 88).
[0902] [0902] Preparation of N2- (3-Amino-2,2-dimethylpropanoyl) -N - [(3R, 4S, 5S) -3-methoxy-1- {(2S) -2 - [(1R, 2R) - 1-methoxy-2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} - 5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide, trifluoroacetic acid salt (# 95)
[0903] [0903] Step 1. Synthesis of 3 - {[(9H-fluoren-9-ylmethoxy) carbonyl] amino} -2,2-dimethylpropanoic acid (# 93). To the 3-amino-2-, 2-dimethylpropanoic acid, hydrochloride salt (250 mg, 1.63 mmol, 1 eq.) In dichloromethane (4 mL, 0.4 M) was added diisopropylethylamine (859 µL, mmol 4 , 88, 3 eq.) Followed by (9H-fluorene-9-ylmethoxy) carbonyl chloride (473 mg, 1.79 mmol, 1.1 eq.). The reaction was stirred for 18 hours and then concentrated in vacuo. The residue was taken up in ethyl acetate (3 ml) and washed with 1 M aqueous hydrochloric acid solution (2 x 1 ml) and with brine. The organic layer was dried over sodium sulfate, filtered, and purified by chromatography on silica gel (Gradient: 0% to 100% ethyl acetate in heptane) to provide # 93 (250 mg, 45%) as an oil. 1H NMR (400 MHz, DMSO-d6) δ 12.22 (s, 1H), 7.89 (d, J = 7.4 Hz, 2H), 7.72 (d, J = 7.4 Hz, 2H ), 7.38-7.44 (m, 2H), 7.27- 7.35 (m, 3H), 4.18-4.30 (m, 3H), 3.16 (d, J = 6 , 2 Hz, 2H), 1.05 (s, 6H).
[0904] [0904] Step 2. Synthesis of N2- (3 - {[(9H-fluoren-9-ylmethoxy) carbonyl] amino} -2,2-dimethylpropanoyl) -N - [(3R, 4S, 5S) -3-methoxy -1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3-oxo-
[0905] [0905] Step 3. Synthesis of N2- (3-amino-2,2-dimethylpropanoyl) -N - [(3R, 4S, 5S) -3- methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1- il} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide, trifluoroacetic acid salt (# 95). For # 94 (86 mg, 0.088 mmol, 1 eq.) In tetrahydrofuran (2 ml, 0.04 M) diethylamine (10 ml) was added. After stirring overnight, the reaction was concentrated in vacuo and the residue was purified by reverse phase chromatography (Method C) to provide # 95 (55 mg, 72%). LC-MS: m / z 757.5 [M + H +], retention time = 0.74 minutes; 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ [8.66 (br d, J = 8 Hz) and 8.92 (br d, J = 9 Hz) , 1H total], [7.91 (br d, J = 8 Hz) and 7.97 (br d, J = 9 Hz), 1H total], [7.78 (d, J = 3.3 Hz) and 7.81 (d, J = 3.1 Hz), total 1H], 7.65-7.74 (br m, 3H), [7.63 (d, J = 3.3 Hz) and 7, 67 (d, J = 3.3 Hz), 1H total], 7.12-7.31 (m, 5H), [5.35-5.42 (m) and 5.45-5.52 (m ), 1H total], [4.44 (dd, J = 9, 9 Hz) and 4.55 (dd, J = 9, 9 Hz), 1H total], 3.17, 3.20, 3.22 and 3.25 (4 s, 6H total), 2.96 and 3.05 (2 br s, 3H total), 1.25 and 1.25 (2 s, 3H total), 1.14 and 1.15 (2 s, 3H total), [1.06 (d, J = 6.6 Hz) and 1.10 (d, J = 6.4 Hz), 3H total], 0.72-0.80 (m , 3H).
[0906] [0906] Preparation of N2- (3-Amino-2,2-dimethylpropanoyl) -N - {(3R, 4S, 5S) -3-methoxy-1- [(2S) -2 - {(1R, 2R) - 1-methoxy-2-methyl-3-oxo-3 - [(2-phenylethyl) amino] propyl} pyrrolidin-1-yl] -5-methyl-1-oxoheptan-4-yl} -N-methyl-L- valinamide, trifluoroacetic acid salt (# 97)
[0907] [0907] Step 1. Synthesis of N2- (3 - {[(9H-fluoren-9-ylmethoxy) carbonyl] amino} -2,2-dimethylpropanoyl) -N - {(3R, 4S, 5S) -3-methoxy -1 - [(2S) -2 - {(1R, 2R) -1-methoxy-2-methyl-3-oxo- 3 - [(2-phenylethyl) amino] propyl} pyrrolidin-1-yl] -5- methyl-1-oxoheptan-4-yl} -N-methyl-L-valinamide (# 96). For # 73 (100 mg, 0.174 mmol, 1 eq.) In dichloromethane (4 ml, 0.04 M) and N, N-dimethylformamide (0.5 ml), # 93 (59.1 mg, 0.174 mmol) was added , 1 eq.), Followed by diisopropylethylamine (92 µL, 0.52 mmol, 3 eq.) And HATU (102 mg, 0.260 mmol, 1.5 eq.). The reaction was stirred for 18 hours and then concentrated in vacuo. The residue was taken up in ethyl acetate (6 ml) and washed with 1 M aqueous hydrochloric acid solution (2 x 2 ml) and with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. Purification by chromatography on silica gel (Gradient: 0% to 50% acetone in heptane) gave # 96 (102 mg, 65%) as a white solid. LC-MS: m / z 896.7 [M + H +], 918.8 [M + Na +], retention time = 1.14 minutes; 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic product signals: δ 7.88 (d, J = 7.4 Hz, 2H), [7.83 (br dd, J = 6.5 Hz) and 8.03 (br dd, J = 6.5 Hz), 1H total], 7.67-7.73 (m, 2H), 7.36-7.48 (m, 3H), 7.22-7.35 (m, 4H), 7.13-7.21 (m, 3H), 6.86-6.96 (m, 1H), [4.44 (dd, J = 8.6, 8.6 Hz) and 4.50 (dd, J = 8.6, 8.6 Hz), total 1H], 3.18, 3.19, 3.26 and 3.29 (4 s, 6H total), 2.96 and 3.11 (2 br s, 3H total), 0.70-0.77 (m, 3H).
[0908] [0908] Step 2. Synthesis of N2- (3-amino-2,2-dimethylpropanoyl) -N - {(3R, 4S, 5S) -3- methoxy-1 - [(2S) -2 - {(1R, 2R) -1-methoxy-2-methyl-3-oxo-3 - [(2-phenylethyl) amino] propyl} pyrrolidin-1-yl] -5-methyl-1-oxoheptan-4-yl} -N-methyl -L-valinamide, trifluoroacetic acid salt (# 97). For # 96 (98 mg, 0.11 mmol, 1 eq.) In tetrahydrofuran (2 ml, 0.04 M) diethylamine (0.5 ml) was added. After stirring overnight, the reaction was concentrated in vacuo and the residue was purified by reverse phase chromatography (Method C) to provide # 97 (58 mg, 68%). LC-MS: m / z 674.4 [M + H +], 696.4 [M + Na +], retention time = 0.74 minutes; HPLC (protocol A): 674.5 [M + H +], retention time = 7.072 minutes; 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ [7.92 (br d, J = 8 Hz) and 7.97 (br d, J = 8 Hz) , 1H total], [7.86 (br dd, J = 6.5 Hz) and 8.07 (br dd, J = 6.5 Hz), 1H total], 7.64-7.74 (br m , 3H), 7.15-7.29 (m, 5H), [4.44 (dd, J = 9, 9 Hz) and 4.50 (dd, J = 9, 9 Hz), total 1H], 3.26 and 3.29 (2 s, 3H total), 3.18 and 3.20 (2 s, 3H total), 2.96 and 3.10 (2 br s, 3H total), 1.24 and 1.25 (2 s, 3H total), 1.14 and 1.16 (2 s, 3H total), 1.02-1.07 (m, 3H), 0.73-0.80 (m, 3H ).
[0909] [0909] Preparation of 2-methyl-L-prolyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1- methoxy-2- methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} - 5-methyl-1- oxoheptan-4-yl] -N-methyl-L-valinamide, trifluoroacetic acid salt (# 98)
[0910] [0910] To a mixture of 1- (tert-butoxycarbonyl) -2-methyl-L-proline (65.1 mg, 0.284 mmol, 1.1 eq.) And # 86 (170 mg, 0.258 mmol, 1 eq. ) in dichloromethane (5 mL, 0.03 M), HATU (0.108 mg, 0.284 mmol, 1.1 eq.) was added followed by diisopropylethylamine (139 µL, 0.800 mmol, 3.1 eq.). After stirring overnight, the reaction mixture was cooled to 0 ° C, dichloromethane (3 ml) was added, followed by the slow addition of trifluoroacetic acid (2 ml). The reaction mixture was stirred at 0 ° C for 5 minutes, allowed to warm up to room temperature and then stirred at room temperature for 30 minutes before being concentrated in vacuo. The residue was azeotroped twice with heptane, diluted with a small amount of dichloromethane and methanol, before being concentrated in vacuo on silica. The residue was purified by chromatography on silica gel (Gradient: 0% to 10% methanol in dichloromethane) and then by reverse phase chromatography (Method C) to provide # 98 (128 mg, 56%) as a solid White. LC-MS: m / z 769.4 [M + H +], retention time = 1.28 minutes; HPLC (Protocol A at 45 ° C) m / z 769.4 [M + H +], retention time = 7.146 minutes (purity> 98%); 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ 9.03-9.15 (br m, 1H), 8.77-8.86 (br m, 1H ), 8.69 - 8.76 (m, 1H), [8.66 (d, J = 8.2 Hz) and 8.92 (d, J = 8.6 Hz), total 1H], [7 , 78 (d, J = 3.1 Hz) and 7.80 (d, J = 3.5 Hz), total 1H], [7.63 (d, J = 3.1 Hz) and 7.67 ( d, J = 3.1 Hz), total 1H], 7.12-7.31 (m, 5H), [5.38 (ddd, J = 11, 8, 4 Hz) and 5.47 (ddd, J = 11, 9, 4 Hz), 1H total], [4.46 (dd, J = 9.4, 9.0 Hz) and 4.55 (dd, J = 9.0, 8.6 Hz) , 1H total], 3.17, 3.20, 3.22 and 3.25 (4 s, 6H total), 2.98 and 3.04 (2 br s, 3H total), [1.06 (d , J = 7.0 Hz) and 1.09 (d, J = 6.6 Hz), total 3H], 0.73-0.80 (m, 3H).
[0911] [0911] Preparation of methyl amino (bicycles [4.2.0] octa-1, 3,5-trien-7-yl) acetate, hydrochloride salt (# 102) &&&&&&&&&&&&&&&& 0 # 2. / &&&&&&&&&&&&&&&&
[0912] [0912] Step 1. Synthesis of ethyl (acetylamino) (bicycles [4.2.0] octa-1,3,5-trien-7-yl) cyanoacetate (# 99). Sodium (464 mg, 20.2 mmol, 1.2 eq.) Was allowed to react with absolute ethanol (40 mL, 0.42 M); To the resulting mixture, ethyl 2- (acetylamino) -2-cyanoacetate (3.44 g, 20.2 mmol, 1.2 eq.) was added. After 20 minutes at 60 ° C, 7-bromobicyclo [4.2.0] octa-1,3,5-triene (3.092 g, 16.89 mmol, 1 eq.) Was added and the reaction mixture was heated to reflux overnight, then filtered and concentrated in vacuo. The residue was diluted with water and extracted with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo to provide a dark oil, which was purified by chromatography on silica gel (Gradient: 0% to 50% ethyl acetate in heptane) to provide # 99 (4.38 g) as a yellow gum. LC - MS: m / z 273.2 [M + H +], retention time = 2.36 minutes.
[0913] [0913] Step 2. Synthesis of (acetylamino) acid (bicycles [4.2.0] octa-1,3,5-trien-7-yl) acetic (# 100). To a mixture of # 99 (4.38 mg, <16.1 mmol, 1 eq.) In methanol
[0914] [0914] Step 3. Amino synthesis of acetic acid (bicycles [4.2.0] octa-1,3,5-trien-7-yl), hydrochloride salt (# 101). A mixture of # 100 (200 mg, 0.912 mmol, 1 eq.) And 6 N aqueous hydrochloric acid (12.3 ml, 73.8 mmol, 81 eq.) Was heated to reflux overnight. The reaction mixture was concentrated in vacuo to provide simple enantiomer # 101 (195 mg) as a yellow solid, which was used in the next step without further purification.
[0915] [0915] Step 4. Synthesis of methyl amino (bicycles [4.2.0] octa-1,3,5-trien-7-yl) acetate, hydrochloride salt (# 102). To a mixture of # 101 (195 mg, <0.913 mmol, 1 eq.) In methanol (20 ml, 0.04 M) was added thionyl chloride (0.666 ml, 9.13 mmol, 10 eq.). After two hours at reflux, the reaction mixture was concentrated in vacuo to provide simple enantiomer # 102 (175 mg, 84% over two steps) as a light colored solid. LC-MS: m / z 192.3 [M + H +], retention time = 0.80 minutes; GC-MS: m / z 192 [M + H +], retention time = 3.206 minutes; 1H NMR (400 MHz, CD3OD) δ 7.24-7.33 (m, 2H), 7.11-7.18 (m, 2H), 4.40 (d, J = 6.9 Hz, 1H) , 3.99-4.05 (m, 1H), 3.78 (s, 3H), 3.46 (dd, J = 14.8, 5.4 Hz, 1H), 3.23 (dd, J = 14.8, 2.5 Hz, 1H).
[0916] [0916] Preparation of (2R, 3R) -3-methoxy-2-methyl-3 - [(2S) -pyrrolidin-2-yl] propanoic acid, hydrochloride salt (# 103) $%% 4 $%
[0917] [0917] To a mixture of # 11 (4.09 g, 14.2 mmol, 1 eq.) In cyclopentyl methyl ether (10 mL, 0.14 M) was added 4 N of a solution of hydrogen chloride in dioxane (37 mL, 100 mmol, 7 eq.). After three hours, the reaction mixture was concentrated in vacuo and azeotroped three times with heptane to provide # 103 (1000 mg, 31%) as a gum, which was used in the next step without further purification. 1H NMR (400 MHz, DMSO-d6) δ 9.92-10.06 (br s, 1H), 8.66 - 8.80 (br s, 1H), 3.89 (dd, J = 5.2 , 4.9 Hz, 1H), 3.43-3.53 (m, 1H), 3.39 (s, 3H), 3.06 - 3.17 (m, 2H), 2.66 (qd, J = 7.1, 4.6 Hz, 1H), 1.71-2.03 (m, 4H), 1.11 (d, J = 7.1 Hz, 3H).
[0918] [0918] Preparation of 2-Methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[1- (bicycle [4.2.0] octa -1,3,5-trien-7-yl) -2-methoxy-2-oxoethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5- methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide, trifluoroacetic acid salt (# 107) and 2-methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) - 2 - [(1R, 2R) -3-
[0919] [0919] Step 1. Syntheses of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -2-methylalanyl-N- [(3R, 4S, 5S) -3-methoxy-5-methyl-1-oxo- 1- (pentafluorophenoxy) heptan-4-yl] -N-methyl-L-valinamide (# 104). For # 32 (4.00 g, 6.56 mmol, 1 eq.) In dichloromethane (20 ml, 0.33 M) and pyridine (1.06 ml, 13.1 mmol, 2 eq.) Was added dropwise drop, pentafluorophenyl trifluoroacetate (2.25 mL, 13.1 mmol, 2 eq.). The reaction mixture was stirred for one hour.
[0920] [0920] To a second flask containing # 32 (360 mg, 0.59 mmol) in dichloromethane (0.6 mL, 1 M) and pyridine (0.095 mL, 1.2 mmol, 2 eq.) Was added dropwise , pentafluorophenyl trifluoroacetate (0.203 mL, 1.18 mmol). This reaction mixture was stirred for 15 minutes.
[0921] [0921] The two reaction mixtures were combined, washed twice with 1 N aqueous hydrochloric acid, dried over sodium sulfate, filtered and concentrated in vacuo. The resulting yellow oil was dissolved in ethyl acetate, pre-adsorbed on silica gel and purified by silica gel chromatography (Gradient: 0% to 40% ethyl acetate in heptane) to provide # 104 (4.6 g, 83%) like a white foam containing some impurities. LC-MS: m / z 798.3 [M + Na +], retention time = 1.23 minutes.
[0922] [0922] Step 2. Synthesis of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -2-methylalanyl-N- [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R , 2R) -2-carboxy-1-methoxypropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 105). To a mixture of # 104 (2.00 g, <2.58 mmol, 1 eq.) In dichloromethane (6 mL, 0.4 M) was added a solution of # 103 (483 mg, 2.16 mmol, 1 eq.) in dichloromethane (2 ml) followed by diisopropylethylamine (1.35 ml, 7.73 mmol, 3 eq.). The reaction mixture was stirred for 16 hours, then adsorbed on silica and purified by silica gel chromatography (Gradient: 0% to 20% methanol in dichloromethane) to provide # 105 (1.67 g, 83%) as a white foam. Fractions containing the desired product with impurities (0.571 g) were collected separately.
[0923] [0923] The above reaction and purification were similarly repeated using # 104 (2.60 g, <3.35 mmol, 1 eq.), # 103 (750 mg, 3.35 mmol, 1 eq.), Dichloromethane (10 mL, 0.3 M) and diisopropylethylamine (1.35 mL, 7.73 mmol, 2.3 eq.) To provide # 105 (2.4 g, 92%) as a brownish foam. Fractions containing the impure product (1.7 g) were combined with the previous impure fractions and purified as described above to provide additional # 105 (1.30 g, quantitative yield for the two reactions over two steps). LC-MS: m / z 779.3 [M + H +], 802.3 [M + Na +], retention time = 1.05 minutes.
[0924] [0924] Step 3. Synthesis of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -2-methylalanyl-N- [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R , 2R) -3 - {[1- (bicycles [4.2.0] octa-1,3,5-trien-7-yl) -2-methoxy-2-oxoethyl] amino} -1-methoxy-2-methyl -3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 106). To a mixture of # 105 (225 mg, 0.289 mmol, 1 eq.) In dichloromethane (15 ml, 0.02 M) and N, N-dimethylformamide (1 ml) was added HATU (136 mg, 0.347 mmol, 1, 2 eq.). After five minutes, a solution of amine # 102 (72.4 mg, 0.318 mmol, 1 eq.) And diisopropylamine (203 µL,
[0925] [0925] Step 4A. Syntheses of 2-methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[1- (bicycle [4.2.0] octa-1, 3,5-trien-7-yl) -2-methoxy-2-oxoethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1 -oxoheptan-4-yl] -N-methyl-L-valinamide, trifluoroacetic acid salt (# 107). According to general procedure A, from # 106 (25 mg, 0.026 mmol, 1 eq.) In dichloromethane (10 ml, 0.003 M) and diethylamine (4 ml), the desired crude material was synthesized, which was purified by reverse phase chromatography (Method C) to provide simple enantiomer # 107 (16 mg, 73%) as a solid. LC-MS: m / z 730.8 [M + H +], retention time = 2.13 minutes; HPLC (Protocol N): retention time = 9.889 minutes.
[0926] [0926] Step 4B. Syntheses of 2-methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3- {[bicycle [4.2.0] octa-1,3,5 -trien-7-yl (carboxy) methyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] - N-methyl-L-valinamide, trifluoroacetic acid salt (# 108). The only # 108 enantiomer (94.5 mg, 57%) was synthesized from # 106 (190 mg, 0.200 mmol), according to a procedure similar to that described for the synthesis of # 41 from # 40. LC-MS: m / z 716.8 [M + H +], retention time = 2.06 minutes; HPLC (Protocol N): retention time = 9.137 minutes.
[0927] [0927] Preparation of 2-methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(1S, 2R) -1- hydroxy- 1-phenylpropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl -L-valinamide (# 112) / $%! 0 $! 1 # 0 "% & / (/ () '(' (
[0928] [0928] Step 1. Synthesis of tert-butyl (2S) -2 - [(1R, 2R) -3 - {[(1S, 2R) -1-hydroxy-1-phenylpropan-2-yl] amino} -1 -methoxy-2-methyl-3-oxopropyl] pyrrolidine-1-carboxylate (# 109). To a solution of # 11 (2.00 g, 6.96 mmol, 1 eq.) In dichloromethane (21 mL, 0.3 M) and N, N-dimethylformamide (3 mL) was added HATU (3.270 mg, 8 , 35 mmol, 1.2 eq.).
[0929] [0929] Step 2. Synthesis of (2R, 3R) -N - [(1S, 2R) -1-hydroxy-1-phenylpropan-2-yl] -3-methoxy-2-methyl-3 - [(2S) -pyrrolidin-2-yl] propanamide, trifluoroacetic acid salt (# 110).
[0930] [0930] Step 3. Synthesis of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -2-methylalanyl-N- [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R , 2R) -3 - {[(1S, 2R) -1-hydroxy-1-phenylpropan-2-yl] amino} -1- methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3- methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 111). According to general procedure D, from # 32 (140 mg, 0.230 mmol, 1 eq.), # 110 (110 mg, 0.253 mmol, 1.1 eq.), Dichloromethane (3 mL, 0.08 M ), N, N-dimethylformamide (0.5 ml), HATU (96.2 mg, 0.253 mmol, 1.1 eq.) And triethylamine (96 µL, 0.69 mmol, 3 eq.) The crude product was synthesized desired, which was purified by silica gel chromatography (Gradient: 0% to 40% acetone in heptane) to provide # 111 (220 mg, 95%). LC-MS: m / z 912.4 [M + H +], 935.4 [M + Na +], retention time = 2.15 minutes; HPLC (Protocol B): m / z 912.5 [M + H +], 934.5 [M + Na +], retention time = 10.138 minutes (purity> 94%); 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ 7.89 (d, J = 7.8 Hz, 2H), 7.66-7.75 (m, 2H), 7.41 (dd, J = 7.4, 7.4 Hz, 2H), 7.12-7.20
[0931] [0931] Step 4. Synthesis of 2-methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3- {[(1S, 2R) -1 -hydroxy-1-phenylpropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] - N-methyl-L-valinamide (# 112). According to general procedure A, from # 111 (210 mg, 0.230 mmol) in dichloromethane (5 ml, 0.05 M) and diethylamine (5 ml), the desired crude material was synthesized, which was purified by chromatography on silica gel (Gradient: 0% to 10% methanol in dichloromethane) to provide a mixture of an oil and a solid. Diethyl ether and heptane were added and the mixture was concentrated in vacuo, providing # 112 (81 mg, 51%) as a white solid. LC-MS: m / z 690.4 [M + H +], retention time = 1.10 minutes; HPLC (protocol A): m / z 690.5 [M + H +], 712.4 [M + Na +], retention time = 7.229 minutes (purity> 90%); 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ [7.62 (br d, J = 8 Hz), 7.88 (br d, J = 8 Hz) , 8.07 (br d, J = 9 Hz) and 8.11 (br d, J = 9 Hz), total 2H], 7.15-7.34 (m, 5H), [5.34 (d , J = 4 Hz) and 5.41 (d, J = 5 Hz), 1H total], 3.18, 3.21, 3.23 and 3.25 (4 s, 6H total), 2.93 and 3.08 (2 br s, total 3H, 1.15, 1.18, 1.21 and 1.25 (4 s, 6H total).
[0932] [0932] Preparation of N, 2-dimethylalanyl-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1- carboxy-2- phenylethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy-1 - [(1S) - 1-methylpropyl] -4-oxobutyl} -N-methyl-L -valinamide, salt of trifluoroacetic acid (115).
[0933] [0933] Step 1. Methyl syntheses N - {(2R, 3R) -3 - [(2S) -1 - {(3R, 4S, 5S) -4 - [{N - [(9H- fluoren-9- ylmethoxy) carbonyl] -L-valyl} (methyl) amino] -3-methoxy-5-methylheptanoyl} pyrrolidin-2-yl] -3-methoxy-2-methylpropanoyl} -L-phenylalaninate (# 113). To a stirring mixture of dimer acid # 5 (12.1 g, 23.0 mM and # 67 (11.5 g, 23.0 mM) in 75 mL of dichloromethane under nitrogen, HATU (10.8 g, 27 , 6 mM) was added followed by Hunig's base (12.1 mL, 69.0 mM). The reaction was allowed to stir at room temperature for 15 hours. The reaction was concentrated to a lower volume, taken up with ethyl acetate and washed with 1 N HCl, twice. The organic layer was then washed with brine, dried over sodium sulfate, filtered, concentrated in vacuo. The residue was then purified by chromatography on silica gel (Gradient: 0 % to 70% acetone in heptanes), providing # 113 (12.3 g, 62%) as a white solid LC-MS (Protocol Q): m / z 855.3 [M + H +], 877.2 [M + Na +], retention time = 2.32 minutes; HPLC (Protocol R): / z 855.5 [M + H +], retention time = 9.596 minutes (purity> 97%).
[0934] [0934] Step 2. Methyl syntheses N - {(2R, 3R) -3-methoxy-3 - [(2S) -1 - {(3R, 4S, 5S) -3- methoxy-5-methyl-4- [methyl (L-valyl) amino] heptanoyl} pyrrolidin-2-yl] -2-methylpropanoyl} -L-phenylalaninate (# 114). In accordance with general procedure A, from # 113 (12 g, 14 mmol, 1 eq.), Dichloromethane (60 ml, 0.24 M) and diethylamine (40 ml, 390 mM) were synthesized # 114 (5 , 9 g, 67%) white / light yellow solid, after purification by chromatography on silica gel (Gradient: 0% to 25% methanol in dichloromethane).
[0935] [0935] Step 3. N, 2-dimethylalanyl-N acid salt syntheses - {(1S, 2R) -4 - {(2S) -2- [(1R, 2R) -3 - {[(1S) - 1-carboxy-2-phenylethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy-1 - [(1S) -1-methylpropyl] -4-oxobutyl} -N-methyl-L-valinamide-trifluoroacetic (# 115). To a stirring mixture of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -N, 2-dimethylalanine (167 mg, 0.493 mM), # 114 (260 mg, 0.411 mM), and HATU (188 mg, 0.493 mM) in 10 ml of dichloromethane, Hunig's base (0.14 ml, 0.82 mM) was added. The reaction was allowed to stir at room temperature for 1 hour and 20 minutes. The reaction was reduced downwards. THF (9 ml) was added to the crude material and to this stirring mixture lithium hydroxide (49.2 mg, 2.06 mM) dissolved in 3 ml of water was added. The reaction was allowed to stir at room temperature for 4 hours. The reaction was concentrated downwards followed by purification by medium pressure reverse phase C18 chromatography (Gradient: 5% to 45% water in acetonitrile with 0.02% TFA in each phase) # 115 (218 mg, 64%) white solid.
[0936] [0936] Preparation of 2-methyl-L-prolyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1- methoxy-3- {[(2S) -1-methoxy-1-oxo-3-phenylpropan-2-yl] amino} -2-methyl-3 oxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl ] -N-methyl-L-valinamide, trifluoroacetic acid salt
[0937] [0937] Step 1. Syntheses of 1- (tert-butoxycarbonyl) -2-methyl-L-prolyl-N - {(1S, 2R) -4- {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-benzyl-2-methoxy-2-oxoethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy-1 - [(1S ) -1-methylpropyl] 4-oxobutyl} -N-methyl-L-valinamide (# 116). To a stirred solution of # 114 (1.02 g, 1.61 mmol, 1.0 eq.) And 1- (tert-butoxycarbonyl) -2-methyl-L-proline (443 mg, 1.93 mmol, 1 , 2 eq.) In 12 ml of dichloromethane, HATU (735 mg, 1.93 mmol, 1.2 eq.) Was added followed by Hunig's base (1.12 ml, 6.45 mmol, 4.0 eq. ). The reaction was allowed to stir at room temperature for 2 hours. The reaction was reduced downwards, diluted with ethyl acetate before being washed with 0.5 N HCl and brine. Organic, where then dried over sodium sulfate, reduced to a small volume, and then reduced to silica. Chromatography on silica was then performed (Gradient: 0% -45% acetone in heptanes) providing # 116 (1.02 g, 74%) as a white solid. LC-MS (Protocol Q): m / z 844.3 [M + H +], 867.2 [M + Na +], retention time = 2.15 minutes.
[0938] [0938] Step 2A. Syntheses of 2-methyl-L-prolyl-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3- {[(1S) -1-benzyl-2-methoxy -2-oxoethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} - 2-methoxy-1 - [(1S) -1-methylpropyl] -4-oxobutyl} -N- methyl-L-valinamide, trifluoroacetic acid salt (# 117). To a stirred solution of # 116 (450 mg, 0.533 mmol, 1.0 eq.) In 7 ml of dichloromethane at 0 ° C, TFA (3 ml, 40 mmol, 70 eq.) Was added. The reaction was allowed to stir at 0 ° C for 5 minutes and then allowed to warm to room temperature while stirring for 20 minutes. The reaction was reduced down, diluted with dichloromethane and a small amount of methanol, before being reduced down over silica. Chromatography on silica was then performed (Gradient: 0% -20% methanol in ethyl acetate) providing # 117 (396 mg, 89%) as a white solid. LC-MS (Protocol Q): m / z 744.5 [M + H +], 767.2 [M + Na +], retention time = 1.40 minutes; HPLC (Protocol A at 45 ° C): m / z 744.5 [M + H +], retention time = 7.149 minutes (purity> 91%). 1 H NMR (400 MHz, DMSO-d6), δ 8.73-9.14 (m), 8.66 (br d), 8.50 (d), 8.22 (d), 7.12- 7.25 (m), 4.67-4.74 (m), 4.41 -4.63 (m), 3.93-4.00 (m), 3.73 (dd), 3.63 (d), 3.46-3.57 (m), 3.38-3.45 (m), 3.26-3.23 (m), 3.22-3.25 (m), 3, 06-3.22 (m), 2.99-3.05 (m), 2.93-2.97 (m), 2.80-2.89 (m), 2.75-2.78 ( m), 2.64-2.67 (m), 2.46-2.50 (m), 2.27-2.43 (m), 2.00-2.26 (m), 1.85 -1.99 (m), 1.70-1.83 (m), 1.52-1.69 (m), 1.33-1.51 (m), 1.18-1.31 (m ), 0.98 -1.07 (m), 0.93-0.97 (m), 0.82-0.92 (m), 0.71-0.78 (m).
[0939] [0939] Step 2B. Syntheses of 2-methyl-L-prolyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3- {[(1S) -1-carboxy-2 -phenylethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide, trifluoroacetic acid salt (# 118). To a stirred solution of # 116 (435 mg, 0.515 mmol), in 4 mL of THF under nitrogen, LiOH (24.7 mg, 1.03 mmol, 2.0 eq.) Dissolved in 2 mL of water was added. The reaction was allowed to stir at room temperature until the indicated saponification of LC-MS methyl ester. The reaction was concentrated in vacuo and then placed under vacuum. The reaction was diluted with dichloromethane and placed under nitrogen. To this stirring mixture, TFA (3 ml, 40.5 mmol, 80 eq.) Was added.
[0940] [0940] Preparation of 2-methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(2S) -1-tert-butoxy- 1-oxo-3-phenylpropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 123). 0; $%
[0941] [0941] Step 1. (2R, 3R) -3 - {(2S) -1 - [(9H-fluoren-9-ylmethoxy) carbonyl] -2-yl} -pyrrolidin-2-yl} -3 -methoxy-2-methylpropanoic (# 119). To a stirred solution of # 11 (2.4 g, 8.4 mmol, 1.0 eq.) In 10 ml of dioxane under nitrogen, 4M HCl in dioxane (20 ml, 80 mM, 10 eq.) Was added . The reaction was allowed to stir at room temperature for 3 hours before being concentrated in vacuo and placed under high vacuum. The crude material was then dissolved with 30 ml of 10% Na2CO3. This solution was then added to a stirred solution of
[0942] [0942] Step 2. Synthesis of tert-butyl N - [(2R, 3R) -3 - {(2S) -1 - [(9H-fluoren-9-ylmethoxy) carbonyl] pyrrolidin-2-yl} -3- methoxy-2-methylpropanoyl] -L-phenylalaninate (# 120). To a stirred solution of tert-butyl L-phenylalaninate, hydrochloride salt (1.67 g, 6.5 mmol, 1.0 eq.) And # 119 (5.9 g, 6.5 mmol, 1.0 eq. .) in 50 mL of dichloromethane and 5 mL of DMF, HATU (2.9 g, 7.9 mmol, 1.2 eq.) was added followed by Hunig's base (5.6 mL, 32 mmol, 5.0 eq.). The reaction was allowed to stir at room temperature for 45 minutes. The reaction was reduced down, diluted with ethyl acetate, washed with 0.5 N HCl and brine before being concentrated to silica. Chromatography on silica was then performed (Gradient: 0% -25% acetone in heptane) providing # 120 (3.14 g, 79%) as a white yellow solid. LC-MS (Protocol Q): m / z 613.1 [M + H +] retention time = 2.37 minutes.
[0943] [0943] Step 3. Synthesis of tert-butyl N - {(2R, 3R) -3-methoxy-2-methyl-3 - [(2S) - pyrrolidin-2-yl] propanoyl} -L-phenylalaninate (# 121 ). To a stirred solution of # 120 (2.87 g, 4.68 mmol, 1.00 eq.) In 20 ml of dichloromethane, diethylamine (10 ml, 95 mM, 20.5 eq.) Was added. The reaction was allowed to stir at room temperature for 2 hours. Another (10 mL, 95 mmol, 20.5 eq.) Of diethylamine was added and the reaction was allowed to stir at room temperature for an additional 3 hours. The reaction was concentrated in vacuo and placed under high vacuum yielding # 121 (1.8 g, quant.) Solid mixture of white yellow oil. LC-MS (Protocol Q): m / z 391.1 [M + H +] retention time = 1.05 minutes.
[0944] [0944] Step 4. Synthesis of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -2-methylalanyl-N- [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R , 2R) -3 - {[(2S) -1-tert-butoxy-1-oxo-3-phenylpropan-2-yl] amino} - 1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl } -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 122). To a stirred solution of # 121 (0.55 g, 1.0 mmol, 1.0 eq.) In mL of dichloromethane and 1 mL of DMF, # 32 (0.62 g, 1.0 mmol, 1.0 eq.) was added followed by HATU (0.42 g, 1.1 mmol, 1.1 eq.) and Hunig's base (0.72 ml, 4.1 mmol, 4.0 eq.). The reaction was allowed to stir at room temperature for approximately 21 hours. The reaction was reduced down, diluted with ethyl acetate, and then washed with 0.5 N HCl and brine. The organic layer was dried over sodium sulfate, filtered, and concentrated to a smaller volume before being concentrated to silica. Chromatography on silica was then performed (Gradient: 0% -40% acetone in heptane) yielding # 122 (0.62 g, 62%) as a white solid. LC-MS (Protocol Q): m / z 982.3 [M + H +] retention time = 2.44 minutes.
[0945] [0945] Step 5. Synthesis of 2-methylalanyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(2S) - 1-tert -butoxy-1-oxo-3-phenylpropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4 -yl] -N-methyl-L-valinamide (# 123). To a stirred mixture of # 122 (600 mg, 0.611 mmol, 1.00 eq.) In 15 ml of dichloromethane, diethylamine (5 ml, 50 mmol, 80 eq.) Was added. The reaction was allowed to stir at room temperature for 3 hours. The reaction was concentrated in vacuo and the mixture was purified by silica chromatography (Gradient: 0% to 40% methanol in dichloromethane) yielding # 123 (0.46 g, 99%) as a solid. LC-MS (Protocol Q1): m / z 760.3 [M + H +] retention time = 0.83 minutes. 1H NMR (400 MHz, CD3OD), δ 7.14-7.30 (m), 4.70-4.78 (m), 4.56-4.64 (m), 4.05-4.19 (m), 3.87
[0946] [0946] Preparation of N - [(2R, 3R) -3 - {(2S) -1 - [(3R, 4S, 5S) -4 - {[N- (3-amino-2,2-dimethylpropanoyl) - L-valyl] (methyl) amino} -3-methoxy-5-methylheptanoyl] pyrrolidin-2-yl} -3-methoxy-2-methylpropanoyl] -L-phenylalaninate (# 126).
[0947] [0947] Step 1. Synthesis of 3 - {[(9H-fluoren-9-ylmethoxy) carbonyl] amino} -2,2-dimethylpropanoic acid (# 124). A solution of 3-amino-2,2-dimethylpropanoic acid hydrochloride (1.0 g, 6.5 mmol, 1.0eq.) In 10 mL of 10% Na2CO3 was added to a solution of 1 - {[(9H-fluoren-9 -ylmethoxy) carbonyl] oxy} pyrrolidine-2,5-dione (2.3 g, 6.5 mmol, 1.0 eq.) in 10 ml of DME. The reaction was allowed to stir at room temperature overnight. The reaction was concentrated to a smaller volume and then washed twice with ether. The aqueous layer was acidified to pH <2 with concentrated HCl and then extracted three times with a solution of 10% methanol 90% dichloromethane. The organics were combined before being washed with 1M HCl and brine. The organic layer was dried over sodium sulfate and concentrated in vacuo yielding # 124 (2.2 g, 98%) as a white solid. LC-MS (Protocol Q1): m / z 362.0 [M + Na +] retention time = 0.89 minutes.
[0948] [0948] Step 2. Synthesis of N - [(2R, 3R) -3 - {(2S) -1 - [(3R, 4S, 5S) -4 - {[N- (3 - {[(9H-fluoren - 9-ylmethoxy) carbonyl] amino} -2,2-dimethylpropanoyl) -L-valyl] (methyl) amino} -3-methoxy-5-methylheptanoyl] pyrrolidin-2-yl} -3-methoxy-2-methylpropanoyl] -L-methyl phenylalaninate (# 125). To a stirred solution of # 114 (200 mg, 0.316 mmol, 1.00 eq.) In 2 ml of dichloromethane, # 124 (107 mg, 0.316 mmol, 1.00 eq.) Was added followed by Hunig's Base (0.167 ml, 0.948 mmol , 3.00 eq.) And HATU (149 mg, 0.379 mmol, 1.20 eq.). The reaction was allowed to stir at room temperature for ~ 12 hours. The reaction was concentrated to a smaller volume, taken in 10 ml of ethyl acetate, and washed twice with 5 ml of 1M HCl, and once with 5 ml of brine. The organic layer was dried over sodium sulfate and decanted. Organics were concentrated in vacuo and the crude material was taken up in dichloromethane. The precipitate was filtered. The organic layer was concentrated in vacuo and the residue was purified by silica chromatography (Gradient: 0% -50% acetone in heptane) yielding # 125 (235 mg, 78%) as a white solid. LC-MS (Protocol Q): m / z 954.2 [M + H +] retention time = 2.28 minutes.
[0949] [0949] Step 3. Synthesis of N - [(2R, 3R) -3 - {(2S) -1 - [(3R, 4S, 5S) -4 - {[N- (3-amino-2,2- dimethylpropanoyl) -L-valyl] (methyl) amino} -3-methoxy-5-methylheptanoyl] pyrrolidin-2-yl} -3-methoxy-2-methylpropanoyl] -L-phenylalaninate methyl (# 126). To a stirred solution of # 125 (235 mg, 0.246 mmol, 1.00 eq.) In 2 ml of THF, (1 ml, 10 mM, 40.6 eq.) Of diethylmine was added. The reaction was allowed to stir at room temperature for 3 hours. The reaction was concentrated in vacuo and the residue was purified by silica chromatography (Gradient: 0% -30% methanol in ethyl acetate) yielding # 126 (101 mg, 56%) as a white solid. LC-MS (Protocol Q): m / z 732.2 [M + H +] retention time = 1.32 minutes. 1H NMR (400 MHz, DMSO-d6), δ 8.51 (dd), 8.28 (d), 7.15-
[0950] [0950] Preparation of N, 2-dimethylanil-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(2S) -1- tert- butoxy-1-oxo-3-phenylpropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4- il] -N-methyl-L-valinamide (# 130).
[0951] [0951] Step 1. Synthesis of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -N, 2-dimethillanyl-N- [(3R, 4S, 5S) -1-tert-butoxy-3-methoxy- 5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 127). To a round bottom flask containing # 6 (4.7 g, 7.9 mmol, 1.0 eq.) And N- [(9H-fluoren-9-ylmethoxy) carbonyl] -N, 2-dimethillanine (3.2 g, 9.4 mmol, 1.2 eq .) and a stir bar under nitrogen, 50 mL of dichloromethane was added followed by HATU (3.6 g, 9.4 mmol, 1.2 eq.) and Hunig's Base (5.5 mL, 32 mmol, 4.0 eq.). The reaction was allowed to stir at room temperature for ~ 12 hours. The reaction was reduced to a smaller volume, taken in ethyl acetate, before being washed with 1 N HCl, and brine. The organics were then dried over sodium sulfate, filtered and then reduced over silica. The residue was purified by silica chromatography (Gradient: 0% -30% acetone in heptane) yielding # 127 (4.2 g, 78%) as a white solid. LC-MS (Protocol Q): m / z 680.2 [M + H +] retention time = 2.52 minutes.
[0952] [0952] Step 2. Synthesis of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -N, 2-dimethillanyl-N- [(2R, 3S, 4S) -1-carboxy-2-methoxy-4- methylhexan-3-yl] -N-methyl-L-valinamide, (# 128). To a stirred solution of # 127 (4.2 g, 6.1 mmol, 1.0 eq.) In 21 ml of dichloromethane under nitrogen, (7 ml, 90 mmol, 10 eq.) Of TFA was added. The reaction was allowed to stir at room temperature for ~ 4 hours. The reaction was concentrated in vacuo, azeotroped once with heptane, and then placed under high vacuum yielding # 128 as a slightly off-white yellow solid (3.8 g, quant.). LC-MS (Protocol Q): m / z 624.2 [M + H +] retention time = 2.01 minutes.
[0953] [0953] Step 3. Synthesis of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -N, 2-dimethillanyl-N- [(3R, 4S, 5S) -1 - {(2S) -2- [ (1R, 2R) -3 - {[(2S) -1-tert-butoxy-1-oxo-3-phenylpropan-2-yl] amino} - 1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1 -yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 129). To a stirred solution of # 128 (1.67 g, 3.1 mmol, 1.0 eq.) In dichloromethane and 2 ml of DMF, # 121 (2.4 g, 3.1 mmol, 1.0 eq.) Was added followed by HATU (1.29 g, 3.39 mmol, 1.1 eq.) and then Hunig's Base (2.2 mL, 12.3 mmol, 4.0 eq.). The reaction was allowed to stir at room temperature for ~ 2 hours. The reaction was reduced, diluted with ethyl acetate before being washed with 0.5 N HCl and brine. Organics were dried over sodium sulfate and then reduced over silica. The residue was purified by silica chromatography (Gradient: 0% -50% acetone in heptanes) yielding # 129 (1.9 g, 62%) as a white solid. LC-MS (Protocol Q): m / z 996.3 [M + H +] retention time = 2.53 minutes.
[0954] [0954] Step 4. Synthesis of N, 2-dimethylanil-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3- {[(2S) -1 -tert-butoxy-1-oxo-3-phenylpropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan -4-yl] -N-methyl-L-valinamide (# 130). To a stirred solution of # 129 (823 mg, 0.826 mmol, 1.00 eq.) In 15 ml of dichloromethane, diethylmine (4 ml, 40 mmol, 50 eq.) Was added. The reaction was allowed to stir at room temperature for ~ 14 ½ hours. The reaction was concentrated in vacuo and azeotroped once with heptanes. The residue was diluted with dichloromethane and a small amount of methanol before being reduced over silica. The residue was purified by silica chromatography (Gradient: 0% -20% methanol in ethyl acetate) yielding # 130 (518 mg, 81%) as a white solid. LC-MS (Protocol Q): m / z 774.3 [M + H +] retention time = 1.48 minutes. HPLC (Protocola A at 25 ° C): m / z 774.5 [M + H +], retention time = 7,733 minutes (purity> 98%). 1H NMR (400 MHz, DMSO-d6), δ 8.36 (d). 8.14 (d), 7.81 (t), 7.14-
[0955] [0955] Preparation of 2-methyl-D-prolyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1- methoxy-3- {[(2S) -1-methoxy-1-oxo-3-phenylpropan-2-yl] amino} -2-methyl-3-oxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4- il] -N-methyl-L-valinamide, trifluoroacetic acid salt (# 131).
[0956] [0956]
[0957] [0957] Step 1. Synthesis of 2-methyl-D-prolyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2- [(1R, 2R) -1-methoxy -3 - {[(2S) -1-methoxy-1-oxo-3-phenylpropan-2-yl] amino} -2-methyl-3-oxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan -4-yl] -N-methyl-L-valinamide, trifluoroacetic acid salt (# 131). To a stirred solution of # 114 (164 mg, 0.259 mmol, 1.0 eq.) And 1- (tert-butoxycarbonyl) -2-methyl-D-proline (71.3 mg, 0.311 mmol, 1.2 eq.) In 4 ml of dichloromethane , HATU (118 mg, 0.311 mmol, 1.2 eq.) Was added followed by Hunig's Base (0.180 mL, 1.04 mmol, 4 eq.). The reaction was allowed to stir at room temperature for ~ 30 minutes. Reaction was reduced. Reaction was taken in
[0958] [0958] Preparation of 2-methyl-L-prolyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(1S, 2R) - 1-hydroxy-1-phenylpropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide, salt of trifluoroacetic acid (# 134).
[0959] [0959] Step 1. Synthesis of N ~ 2 ~ - [(9H-fluoren-9-ylmethoxy) carbonyl] -N - [(3R, 4S, 5S) -1- {(2S) -2 - [(1R, 2R) -3 - {[(1S, 2R) -1-hydroxy-1-phenylpropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 132). To a vial containing # @ 5 (1.14 g, 2.17 mmol, 1.0 eq.), 10 ml of dichloromethane was added followed by Hunig's Base (1.15 ml, 6.52 mmol, 3.0 eq.), HATU (1.02 g, 2.61 mmol, 1.2 eq.), And # 110 (0.776 g, 2.17 mmol, 1.0 eq.).
[0960] [0960] Step 2. Synthesis of N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(1S, 2R) -1-hydroxy- 1 -phenylpropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl- L-valinamide (# 133). To a stirred solution of # 132 (1.33 g, 1.60 mmol, 1.0 eq) in 10 ml of diethylmin THF (5 ml, 50 mM, 31.3 eq) was added. The reaction was allowed to stir at room temperature for 4 hours.
[0961] [0961] Step 3. Synthesis of 2-methyl-L-prolyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3- {[(1S, 2R) -1-hydroxy-1-phenylpropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4 -yl] -N-methyl-L-valinamide, trifluoroacetic acid salt (# 134). HATU (151 mg, 0.398 mmol, 1.2 eq), # 133 (201 mg, 0.332 mmol, 1.0 eq.) And 1- (tert-butoxycarbonyl) -2-methyl-L-proline (91.3 mg, 0.398 mM, 1.2 eq .) were combined in a round-bottom flask containing a stir bar under nitrogen. 5 mL of dichloromethane was added followed by Base
[0962] [0962] Preparation of N, 2-dimethylanil-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1- benzyl-2- (methylmino) -2-oxoethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy-1 - [(1S) -1-methylpropyl] -4-oxobutile} -N-methyl-L-valinamide, trifluoroacetic acid salt (# 140), N, 2-dimethylanil-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -2- amino-1-benzyl-2-oxoethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy- 1 - [(1S ) -1-methylpropyl] -4-oxobutyl} -N-methyl-L-valinamide, trifluoroacetic acid salt (# 141), N, 2-dimethylanil-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-benzyl-2-oxo-2- (propylamino) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin- 1-yl} -2-methoxy-1 - [(1S) - 1-methylpropyl] -4-oxobutyl} -N-methyl-L-valinamide, trifluoroacetic acid salt (# 142), N, 2-dimethylanil-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-benzyl-2- (diethylmino) -2- oxoethyl] amino} -1 -methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy-1 - [(1S) -1- methylpropyl] -4-oxobutyl} -N-methyl-L-valinamide, acid salt trifluoroacetic o (# 143), and
[0963] [0963] Step 1. Synthesis of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -N, 2-dimethillanyl-N-
[0964] [0964] Step 2. Synthesis of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -N, 2-dimethillanyl-N- [(3R, 4S, 5S) -1 - {(2S) -2- [ (1R, 2R) -2-carboxy-1-methoxypropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 136). To a stirred solution of 4M HCl in dioxane (10 mL, 25 mmol, 3.7 eq.) In 10 mL of dioxane # 11 (2.31 g, 8.05 mmol, 1.2 eq.) Was added. The reaction was allowed to stir at room temperature for 6 hours. The reaction was concentrated in vacuo to produce a yellow gum. A solution of # 135 (5.3 g, 6.7 mmol, 1.0 eq.) In 30 ml of dichloromethane was added to the previous residue followed by Hunig's Base (3.5 ml, 20 mmol, 3 eq.).
[0965] [0965] Step 3. Synthesis of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -N, 2-dimethillanyl-N- [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3-oxo-3- (pentafluorophenoxy) propyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N -methyl-L-valinamide (# 137). Pentafluorophenyl 3,3,3-trifluoropropanoate (1.3 mL, 7.1 mmol, 2.0 eq.) Was added to a solution of # 136 (2.8 g, 3.5 mmol, 1.0 eq.) In 30 mL of dichloromethane followed by the addition of pyridine ( 0.85 mL, 10.6 mM). The reaction was allowed to stir at room temperature for 2 hours. The reaction was concentrated in vacuo, and the residue was purified by silica chromatography (Gradient: 0% -70% acetone in heptane) yielding # 137 (3.1 g, 92%) as a white powder. LC-MS (Protocol Q1): m / z 959.2 [M + H +] retention time = 1.28 minutes.
[0966] [0966] Step 4. Synthesis of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -N, 2-dimethillanyl-N- [(3R, 4S, 5S) -1 - {(2S) -2- [ (1R, 2R) -3 - {[(1S) -1-carboxy-2-phenylethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5- methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 138). To a stirred solution of # 137 (493 mg, 0.514 mmol, 1.0 eq.) In 4 mL of DMF, L-phenylalanine (84.9 mg, 0.514 mmol, 1.0 eq) was added followed by Hunig's Base (0.27 mL, 1.54 mmol , 3.0 eq.). The reaction was allowed to stir at room temperature for ~ 12 hours. The reaction was concentrated in vacuo and the residue was purified by silica chromatography (Gradient: 0% -100% ethyl acetate in heptane) yielding # 138 (200 mg, 41%) as a white foam. LC-MS (Protocol Q1): m / z 940.3 [M + H +] retention time = 1.08 minutes.
[0967] [0967] Step 5. Synthesis of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -N, 2-dimethillanyl-N- [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3-oxo-3 - {[(2S) -1-oxo-1- (pentafluorophenoxy) -3-phenylpropan-2-yl] amino} propyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 139). To a stirred solution of # 138 (200 mg, 0.213 mmol, 1.0 eq.) In 5 ml of dichloromethane, pentafluorophenyl 3,3,3-trifluoropropanoate (126 mg, 0.426 mM, 2.0 eq.) Was added followed by pyridine ( 0.051 mL, 0.64 mmol, 3.0 eq.). The reaction was allowed to stir at room temperature for ~ 12 hours. The reaction was concentrated in vacuo and the residue was purified by silica chromatography (Gradient: 0% -100% ethyl acetate in heptanes) yielding # 139 (174 mg, 74%) as a yellow oil. LC-MS (Protocol Q1): m / z 1128 [M + Na +] retention time = 1.23 minutes.
[0968] [0968] Step 6A. Synthesis of N, 2-dimethylanil-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) - 1-benzyl-2- (methylmino) -2-oxoethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy-1 - [(1S) -1-methylpropyl] -4-oxobutyl} -N- methyl-L-valinamide, trifluoroacetic acid salt (# 140). To a stirred solution of # 139 (20 mg, 0.018 mmol, 1.0 eq.) In 1mL of methyl THF (1M in THF, 0.18 mL, 0.18 mmol, 10 eq.) Was added, and the mixture was stirred at room temperature for 3 hours. The reaction was reduced, diluted with dmso, and subjected to purification (Method J *). Fractions were collected and concentrated in vacuo to give # 140 (4.0 mg, 30%) as a white solid. LC-MS (Protocol Q1): m / z 731.2 [M + H +], retention time = 0.70 minutes. 1H NMR (400 MHz, methanol-d4), 7.30-7.41 (m), 4.71-4.78 (m), 4.58-4.69 (m), 4.04-4.15 (m), 3.86-3.98 (m), 3.73-3.78 ( m), 3.61-3.70 (m), 3.50-3.58 (m), 3.32-
[0969] [0969] Step 6B. Synthesis of N, 2-dimethylanil-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) - 2-amino-1-benzyl-2 -oxoethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy-1 - [(1S) -1-methylpropyl] -4-oxobutyl} -N-methyl- L-valinamide, salt of trifluoroacetic acid (# 141). Following the same procedure as # 140 using # 139 (20 mg, 0.018 mmol, 1.0 eq.), Ammonia solution (7M in methanol, 0.026 mL, 0.18 mmol, 10eq.) And purification (Method J *), # 141 ( 3.0 mg, 20%) was obtained as a white solid. LC-MS (Protocol Q): m / z 717.2 [M + H +], retention time = 0.79 minutes. 1 H NMR (400 MHz, methanol-d4), 7.22-7.30 (m), 7.14-7.21 (m), 4.57-4.4.80 (m), 4.02-
[0970] [0970] Step 6C. Synthesis of N, 2-dimethylanil-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) - 1-benzyl-2-oxo-2 - (propylamino) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} - 2-methoxy-1 - [(1S) -1-methylpropyl] -4-oxobutyl} -N -methyl-L-valinamide, trifluoroacetic acid salt (# 142). Following the same procedure as # 140 using # 139 (20 mg, 0.018 mmol, 1.0 eq.) N-propylamine (1M in THF, 0.18 mL, 0.18 mmol, 10. eq) and purification (Method J *), # 142 ( 3.0 mg, 20%) was obtained as a white solid. LC-MS (Protocol Q): m / z 759.2 [M + H +], retention time = 0.74 minutes. 1H NMR (400 MHz, methanol-d4), 7.15-7.29 (m), 4.71-4.79 (m), 4.52-4.68 (m), 4.04-4.17 (m),
[0971] [0971] Step 6D. Synthesis of N, 2-dimethylanil-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) - 1-benzyl-2- (diethylmino) -2-oxoethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy-1 - [(1S) -1-methylpropyl] -4-oxobutyl} -N- methyl-L-valinamide, trifluoroacetic acid salt (# 143). Following the same procedure as # 140 using # 139 (20 mg, 0.018 mmol, 1.0 eq.) Diethylmine (1M in THF, 0.18 mL, 0.18 mmol, 10 eq.) And purification (Method J *), # 143 (4.0 mg , 30%) was obtained as a white solid. LC-MS (Protocol Q): m / z 773.3 [M + H +], retention time = 0.77 minutes. 1H NMR (400 MHz, methanol-d4), 7.16-7.33 (m), 5.10-5.17 (m), 4.96-5.07 (m), 4.68-4.75 (m),
[0972] [0972] Step 6E. Synthesis of N, 2-dimethylanil-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) - 1-benzyl-2- (tert- butylamino) -2-oxoethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy-1 - [(1S) -1-methylpropyl] -4-oxobutyl} - N-methyl-L-valinamide, salt of trifluoroacetic acid # 144. Following the same procedure as # 140 using # 139 (20 mg, 0.018 mmol, 1.0eq.) Tert-butylmine (1M in THF, 0.18 mL, 0.18 mmol, 10 eq.) And purification (Method J *), # 144 ( 3.4 mg, 24%) was obtained as a white solid. LC-MS (Protocol Q1): m / z 773.3 [M + H +], retention time = 0.74 minutes. 1H NMR (400 MHz, DMSO-d6), δ 8.21 (d), 8.03-7.98 (m), 7.92 (d), 7.81-7.62 (m), 7.46-7.16 (m), 4.83-4.69 (m), 4.68-4.56 (m), 4.21-4.07 (m), 3.92-3.86 (m), 3.83-3.80 (m), 3.74-
[0973] [0973] Preparation of N, 2-dimethylanil-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3- {[(1S, 2R) -1- hydroxy-1-phenylpropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-valinamide, salt of trifluoroacetic acid (# 145).
[0974] [0974] Step 1. Synthesis of N, 2-dimethylanil-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3- {[(1R, 2S) -2 -hydroxy-1-methyl-2-phenylethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy-1 - [(1S) -1-methylpropyl] -4 -oxobutyl} -N-methyl-L-valinamide, trifluoroacetic acid salt (# 145). To a stirred solution of # 137 (300 mg, 0.313 mmol, 1.0 eq.) In 3 mL of DMF, (1S, 2R) -2-amino-1-phenylpropan-1-ol (54.8 mg, 0.344 mmol, 1.1 eq .) was added followed by Hunig's Base (0.164 mL, 0.939 mmol, 3.0 eq). The reaction was allowed to stir at room temperature for ~ 12 hours. 20% DMF piperidine solution (1 mL, 2.2 mmol, 7.0 eq.) Was then added and the reaction was allowed to stir at room temperature for 2 hours. Purification (Method J *) followed by concentration of appropriate test tubes produced # 145 (190 mg, 74%) as a white powder. LC-MS (Protocol Q): m / z 704.3 [M + H +], retention time = 0.67 minutes. 1H NMR (400 MHz, CD3OD), δ 7.97 (d), 7.73 (d), 7.37-7.41 (m), 7.27-7.36 (m), 7.19-7.25 (m), 4.70- 4.75 (m), 4.58- 4.63 (m), 4.49-4.54 (m), 4.14-
[0975] [0975] Preparation of 3-methyl-D-isovalyl-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1- benzyl- 2-methoxy-2-oxoethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy- 1 - [(1S) -1-methylpropyl] -4-oxobutile} -N-methyl-L-valinamide (# 146), 3-methyl-L-isovalyl-N- {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[ (1S) -1-benzyl-2-methoxy-2-oxoethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy-1 - [(1S) -1 -methylpropyl] -4-oxobutyl} -N-methyl-L-valinamide (# 147), L-isovalyl-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) - 3 - {[(1S) -1-benzyl-2-methoxy-2-oxoethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy-1 - [( 1S) - 1-methylpropyl] -4-oxobutyl} -N-methyl-L-valinamide (# 148), and D-isovalyl-N - {(1S, 2R) -4 - {(2S) - 2 - [( 1R, 2R) -3 - {[(1S) -1-benzyl-2-methoxy-2-oxoethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy -1 - [(1S) -1-methylpropyl] -4-oxobutyl} -N-methyl-L-valinamide (# 149).
[0976] [0976] Step 1A. Synthesis of 3-methyl-D-isovalyl-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3- {[(1S) -1-benzyl-2-methoxy -2-oxoethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -
[0977] [0977] Step 1B. Synthesis of 3-methyl-L-isovalyl-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3- {[(1S) -1-benzyl-2-methoxy -2-oxoethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} - 2-methoxy-1 - [(1S) -1-methylpropyl] -4-oxobutyl} -N- methyl-L-valinamide (# 147). A solution of # 114 (224 mg, 0.354 mmol, 1.0 eq.) In 2 ml of dichloromethane was added to a solution of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -3-methyl-L-isovaline ( 125 mg,
[0978] [0978] Step 1C. Synthesis of L-isovalyl-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1- benzyl-2-methoxy-2-oxoethyl ] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy- 1 - [(1S) -1-methylpropyl] -4-oxobutyl} -N-methyl-L- valinamide (# 148). A solution of # 114 (447 mg, 0.707 mmol, 1.0 eq.) In 2 ml of dichloromethane was added to a solution of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -L-isovaline (240 mg, 0.707 mmol,
[0979] [0979] Step 1D. Synthesis of D-isovalyl-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1- benzyl-2-methoxy-2-oxoethyl ] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy- 1 - [(1S) -1-methylpropyl] -4-oxobutyl} -N-methyl-L- valinamide (# 149). A solution of # 114 (447 mg, 0.707 mmol, 1.0 eq.) In 2 ml of dichloromethane was added to a solution of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -D-isovaline (240 mg, 0.707 mmol, 1.0 eq.) in 4 ml of dichloromethane. Hunig's base (0.373 mL, 2.12 mmol, 3 eq.) Was added followed by HATU (332 mg, 0.425 mmol, 1.2 eq.). The reaction was allowed to stir at room temperature for 12 hours. The reaction was concentrated in vacuo and then taken up in ethyl acetate before being washed twice with 1M HCl and once with brine. The organic layer was dried over sodium sulfate and decanted. The organic solvent was removed in a genevac. THF (4 ml) was added followed by diethylmine (2 ml, 19 mmol, 26.9 eq.). The reaction was allowed to stir for ~ 12 hours. Reaction was concentrated using a genevac followed by silica chromatography (Gradient: 0% -30% methanol in ethyl acetate) yielding # 149 (154 mg, 30%) as a solid. LC-MS (Protocol Q): m / z 732.0 [M + H +] retention time = 1.24 minutes. 1H NMR (400 MHz, DMSO-d6), δ 8.55 (d),
[0980] [0980] Preparation of 1,2-dimethyl-L-prolyl-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1- carboxy-2-phenylethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy-1 - [(1S) - 1-methylpropyl] -4-oxobutyl} -N -methyl-L-valinamide (# 151).
[0981] [0981] Step 1. Synthesis of 1,2-dimethyl-L-proline (# 150). One bottle containing 2-methyl-L-proline (1.0 g, 7.7 mmol, 1.0 eq.), 40 ml of methanol, Formaldehyde 37% by weight in water (2.1 ml, 77 mmol, 10 eq.), And 10% by weight of palladium on carbon (313 mg, 2.94 mmol, 0.38 eq.) was placed on a stirrer and allowed to stir under 40 psi of hydrogen for ~ 12 hours. Hydrogen was removed and the reaction was filtered through a pad of celite that was washed with a solution of 50% methanol 50% dichloromethane. Residue was concentrated in vacuo yielding # 150 (1.1 g, 100%) as an off-white light black colored solid. LC-MS (Protocol Q): m / z 144.0 [M + H +] retention time = 0.17 minutes.
[0982] [0982] Step 2. Synthesis of 1,2-dimethyl-L-prolyl-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3- {[(1S) -1-carboxy-2-phenylethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2- methoxy-1 - [(1S) -1-methylpropyl] -4-oxobutyl } -N-methyl-L-valinamide (# 151). To a stirred mixture of # 114 (125 mg, 0.198 mmol, 1.0 eq), # 150 (37 mg, 0.26 mmol, 1.3 eq.), And HATU (98 mg, 0.26 mmol, 1.3 eq.) In 5 ml of dichloromethane , Hunig's base (0.14 mL, 0.80 mmol, 4.1 eq.) Was added. The reaction was allowed to stir at room temperature for 1 hour. Reaction was concentrated in vacuo. THF (6 mL) was added to the crude material. To this stirring mixture LiOH (14 mg, 0.59 mmol, 3.0 eq) dissolved in 2 ml of water was added. Reaction was allowed to stir at room temperature for 90 minutes. Reaction was concentrated in vacuo and residue was purified by medium pressure reverse phase C18 chromatography (Gradient: 5% to 40% acetonitrile in water with 0.02% TFA in each phase) # 151 (147 mg, 69%) as a white solid. LC-MS (Protocol Q): m / z 744.3 [M + H +], retention time = 1.19 minutes; HPLC (Protocola A at 45 ° C): m / z 744.4 [M + H +], retention time = 6,631 minutes (purity> 98%). 1H NMR (400 MHz, DMSO-d6), δ 9.57-9.71 (m),
[0983] [0983] Preparation of 1,2-dimethyl-D-prolyl-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1- carboxy-2-phenylethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-methoxy-1 - [(1S) - 1-methylpropyl] -4-oxobutyl} -N -methyl-L-valinamide (# 153).
[0984] [0984] Step 1. Synthesis of 1,2-dimethyl-D-proline (# 152). To a parr bottle containing 2-methyl-D-proline (432 mg, 3.34 mmol, 1.0 eq.), Formaldehyde 37% by weight in water (1.0 mL, 37 mM, 11 eq.), 3.5 mL of methanol and 1 mL of water, 10 wt% palladium on carbon (108 mg, 0.304 mmol, 0.304 eq.) was added. The flask was placed on a parr shaker and allowed to stir under 30 psi of hydrogen for ~ 48 hours. Hydrogen was removed and the reaction was washed through a celite pad, which was subsequently washed with methanol. The organics were concentrated in vacuo and then azeotroped with toluene providing # 152 (517 mg, 100%) as a solid. 1H NMR (400 MHz, methanol-d4): δ [3.61-3.56 (m, 1H), 3.07-2.96 (m, 1H), 2.68 (br s, 3H), 2.34-2.22 (m, 1H), 2.01- 1.88 (m, 1H), 1.87-
[0985] [0985] Step 2. Synthesis of 1,2-dimethyl-D-prolyl-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3- {[(1S) -1-carboxy-2-phenylethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2- methoxy-1 - [(1S) -1-methylpropyl] -4-oxobutyl } -N-methyl-L-valinamide (# 153). To a stirred mixture of # 114 (240 mg, 0.379 mmol, 1.0 eq.), # 152 (71 mg, 0.49 mmol, 1.3 eq.), And HATU (188 mg, 0.49 mmol, 1.3 eq.) In 10 mL of dichloromethane, Hunig's Base (0.27 mL, 4.1 mM, 4.1 eq.) was added. The reaction was allowed to stir at room temperature for 1 hour. Reaction was concentrated in vacuo. THF (6 mL) was added to the crude material. To this stirring mixture LiOH (36 mg, 1.5 mmol, 4 eq.) Dissolved in 2 ml of water was added. Reaction was allowed to stir at room temperature for 1 hour. The reaction was concentrated in vacuo and the residue was purified by medium pressure reverse phase C18 chromatography (Gradient: 5% to 40% acetonitrile in water with 0.02% TFA in each phase) # 153 (220 mg, 78%) as a white solid. LC-MS (Protocol Q): m / z 744.8 [M + H +], retention time = 1.16 minutes; HPLC (Protocola A at 45 ° C): / z 744.4 [M + H +], retention time = 6.713 minutes (purity> 98%). 1H NMR (400 MHz, DMSO-d6), δ 9.72-9.85 (m), 8.65 (t),
[0986] [0986] Preparation of N ~ 2 ~ - [2,2-dimethyl-3- (methylmino) propanoyl] -N - {(1S, 2R) -2- methoxy-4 - {(2S) -2 - [(1R , 2R) -1-methoxy-2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1 -il} -1 - [(1S) -1-methylpropyl] -4-oxobutyl} -N-methyl-L-valinamide, trifluoroacetic acid salt (# 154).
[0987] [0987] Step 1. Synthesis of N ~ 2 ~ - [2,2-dimethyl-3- (methylmino) propanoyl] -N - {(1S, 2R) -2- methoxy-4 - {(2S) -2- [(1R, 2R) -1-methoxy-2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} -1 - [(1S) -1-methylpropyl] -4-oxobutyl} -N-methyl-L-valinamide, trifluoroacetic acid salt (# 154). To a vial containing # 50 (100 mg,
[0988] [0988] Preparation of N - {(2R, 3R) -3 - [(2S) -1 - {(3R, 4S, 5S) -4 - [{N- [2,2-dimethyl-3- (methylmino) propanoyl] -L-valyl} (methyl) amino] -3-methoxy-5-methylheptanoyl} pyrrolidin-2-yl] -3-methoxy-2-methylpropanoyl} -L-phenylalaninate, trifluoroacetic acid salt (# 155 ).
[0989] [0989] Step 1. Synthesis of N - {(2R, 3R) -3 - [(2S) -1 - {(3R, 4S, 5S) -4 - [{N- [2,2-dimethyl-3- (methylmino) propanoyl] -L-valyl} (methyl) amino] -3-methoxy-5-methylheptanoyl} pyrrolidin-2-yl] -3-methyloxy-2-methylpropanoyl} -L-methylphenylalaninate, trifluoroacetic acid salt (# 155).
[0990] [0990] Preparation of N - {(2R, 3R) -3-methoxy-3 - [(2S) -1 - {(3R, 4S, 5S) -3-methoxy-5-methyl- 4- [methyl (N - {{((2S) -2-methylpiperidin-2-yl] carbonyl} -L-valyl) amino] heptanoyl} pyrrolidin-2-yl] -2-methylpropanoyl} -L-phenylalaninate methyl, salt of trifluoroacetic acid (# 158) and N- {(2R, 3R) -3-methoxy-3 - [(2S) -1 - {(3R, 4S, 5S) -3-methoxy-5-methyl-4- [methyl (N- { [(2R) -2- methylpiperidin-2-yl] carbonyl} -L-valyl) amino] heptanoyl} pyrrolidin-2-yl] -2-methylpropanoyl} -L-phenylalaninate, trifluoroacetic acid salt (# 159)
[0991] [0991] Step 1. (Synthesis of (2S) -1- (tert-butoxycarbonyl) -2-methylpiperidine-2-carboxylic acid (# 156) and (2R) -1- (tert-butoxycarbonyl) -2-methylpiperidine -2-carboxylic acid (# 157). 1- (tert-butoxycarbonyl) -2-methylpiperidine-2-carboxylic acid (500 mg, 2.06 mmol, 1 eq.) Was separated by supercritical fluid chromatography (Column: Chiralcel OJ-H , 250 x 21 mm; Eluent: 90:10 carbon dioxide / ethanol; Flow rate: 65 g / min; to give the corresponding enanciomers. The first elution peak (retention time = 1.57 minutes) was isolated to give # 156 as a gum (140 mg, 28%) (stereochemistry arbitrarily assigned as the S enanciomer). 1H NMR (400 MHz, CDCl3) δ 3.83-3.90 (m, 1H), 2.93-3.01 (m, 1H), 1.87- 1.97 (m, 1H),
[0992] [0992] Step 2A. Synthesis of N - {(2R, 3R) -3-methoxy-3 - [(2S) -1 - {(3R, 4S, 5S) -3-methoxy-5-methyl-4- [methyl (N - {[ (2S) -2-methylpiperidin-2-yl] carbonyl} -L valyl) amino] heptanoyl} pyrrolidin-2-yl] -2-methylpropanoyl} -L-phenylalaninate methyl, salt of trifluoroacetic acid (# 158). To a solution of # 156 (8.3 mg, 0.034 mmol, 1 eq.) In dichloromethane (0.3 mL) and N, N-dimethylformamide (0.05 mL), N, N-diisopropylethylmine (0.018 mL, 0.102 mmol, 3 eq) was added .), followed by HATU (16.1 mg, 0.041 mmol, 1.2 eq.). The reaction was stirred for 15 minutes and # 114 (23.4 mg, 0.037 mmol,
[0993] [0993] Step 2B. Synthesis of N - {(2R, 3R) -3-methoxy-3 - [(2S) -1 - {(3R, 4S, 5S) -3-methoxy-5-methyl-4- [methyl (N - {[ (2R) -2-methylpiperidin-2-yl] carbonyl} -L-valyl) amino] heptanoyl} pyrrolidin-2-yl] -2-methylpropanoyl} -L-phenylalaninate methyl, salt of trifluoroacetic acid (# 159). To a solution of # 157 (7.8 mg, 0.032 mmol, 1 eq.) In dichloromethane (0.3 ml) and N, N-dimethylformamide (0.05 ml), was added N, N-
[0994] [0994] Preparation of N - {(2R, 3R) -3-methoxy-3 - [(2S) -1 - {(3R, 4S, 5S) -3-methoxy-5-methyl- 4- [methyl (N - {[(2S) -2-methylpiperidin-2-yl] carbonyl} -L-valyl) amino] heptanoyl} pyrrolidin-2-yl] -2- methylpropanoyl} -L-phenylalanine, trifluoroacetic acid salt (# 162) and N - {(2R, 3R) -3- methoxy-3 - [(2S) -1 - {(3R, 4S, 5S) -3-methoxy-5-methyl-4- [methyl (N - {[( 2R) -2-methylpiperidin-2-yl] carbonyl} -L-valyl) amino] heptanoyl} pyrrolidin-2-yl] -2-methylpropanoyl} -L-phenylalanine, trifluoroacetic acid salt. (# 163).
[0995] [0995] Step 1A. Synthesis of N - {(2R, 3R) -3 - [(2S) -1 - {(3R, 4S, 5S) -4 - [(N - {[(2S) -1- (tert-butoxycarbonyl) -2 -methylpiperidin-2-yl] carbonyl} -L-valyl) (methyl) amino] -3-methoxy-5-methylheptanoyl} pyrrolidin-2-yl] -3-methoxy-2-methylpropanoyl} -L-phenylalaninate ( # 160). To a solution of # 156 (106 mg, 0.436 mmol, 1 eq.) In dichloromethane (3 mL) and N, N-dimethylformamide (0.5 mL) was added diisopropylethylmine (0.228 mL,
[0996] [0996] Step 1B. Synthesis of N - {(2R, 3R) -3 - [(2S) -1 - {(3R, 4S, 5S) -4 - [(N - {[(2R) -1- (tert-butoxycarbonyl) -2 -methylpiperidin-2-yl] carbonyl} -L-valyl) (methyl) amino] -3-methoxy-5-methylheptanoyl} pyrrolidin-2-yl] -3-methoxy-2-methylpropanoyl} -L-phenylalaninate ( # 161). To a solution of # 157 (109 mg, 0.448 mmol, 1 eq.) In dichloromethane (3 mL) and N, N-dimethylformamide (0.5 mL), was added diisopropylethylmine (0.234 mL,
[0997] [0997] Step 2A. Synthesis of N - {(2R, 3R) -3-methoxy-3 - [(2S) -1 - {(3R, 4S, 5S) -3-methoxy-5-methyl-4- [methyl (N - {[ (2S) -2-methylpiperidin-2-yl] carbonyl} -L-valyl) amino] heptanoyl} pyrrolidin-2-yl] -2-methylpropanoyl} -L-phenylalanine, trifluoroacetic acid salt (# 162). To a solution of # 160 (145 mg, 0.169 mmol, 1 eq.) In tetrahydrofuran (1.25 ml) was added lithium hydroxide (8 mg, 0.338 mmol, 2 eq.) Dissolved in water (0.75 ml). The reaction was stirred at room temperature for 2 hours and evaporated to dryness in vacuo. The residue was dissolved in dichloromethane (2.5 ml) and trifluoroacetic acid (1 ml) was added. The reaction was stirred for 30 minutes, concentrated in vacuo and purified by medium pressure reverse phase C18 chromatography (Gradient: 0% to 100% acetonitrile in water with 0.02% TFA in each phase) to yield the title compound # 162 (145 mg, quantitative) as a white solid. HPLC (Protocol U): m / z 744.5 [M + H +], retention time =
[0998] [0998] Step 2B. Synthesis of N - {(2R, 3R) -3-methoxy-3 - [(2S) -1 - {(3R, 4S, 5S) -3-methoxy-5-methyl-4- [methyl (N - {[ (2R) -2-methylpiperidin-2-yl] carbonyl} -L-valyl) amino] heptanoyl} pyrrolidin-2-yl] -2-methylpropanoyl} -L-phenylalanine, trifluoroacetic acid salt (# 163). Compound # 161 (185 mg, 0.216 mmol, 1 eq.) Was converted to the crude title compound # 163, using the procedure described for the preparation of # 162. The crude material was purified by medium pressure reverse phase C18 chromatography (Gradient: 0% to 85% acetonitrile in water with 0.02% TFA in each phase) to yield # 163 (127 mg, 68%) as a white solid . HPLC (Protocol U): m / z 744.5 [M + H +], retention time = 7,077 minutes (purity = 98%). 1H NMR (400 MHz, DMSO-d6). δ 8.79-8.99 (m), 8.36-8.49 (m), 8.12-8.17 (m),
[0999] [0999] Preparation of N - {(2R, 3R) -3 - [(2S) -1 - {(3R, 4S, 5S) -4 - [(N - {[(3R) -3- fluoropyrrolidin-3- yl] carbonyl} -L-valyl) (methyl) amino] -3-methoxy-5-methylheptanoyl} pyrrolidin-2-yl] -3-methoxy-2-methylpropanoyl} -L-phenylalaninate methyl, salt of trifluoroacetic acid ( # 172) and N - {(2R, 3R) -3 - [(2S) -1 - {(3R, 4S, 5S) -4 - [(N - {[(3R) -3-fluoropyrrolidin-3-yl ] carbonyl} -L-valyl) (methyl) amino] -3-methoxy-5-methylheptanoyl} pyrrolidin-2-yl] -3-methoxy-2-methylpropanoyl} -L-phenylalaninate methyl, salt of trifluoroacetic acid (# 173).
[01000] [01000] Step 1. Synthesis of methyl (3R) -1-benzyl-3-fluoropyrrolidine-3-carboxylate (# 164) and methyl (3S) -1-benzyl-3-fluoropyrrolidine-3-carboxylate (# 165 ). (Known methyl 1-benzyl-3-fluoropyrrolidine-3-carboxylate (3900 mg, 16.4 mmol, 1 eq.) Was separated by supercritical fluid chromatography (Column: Chiralpak IC, 250 x 21 mm; Eluent: 95: 5 dioxide carbon / propanol; Flow Rate: 65 g / min; to give the corresponding enanciomers.The first elution peak (retention time =
[01001] [01001] Step 2A. Synthesis of 1-tert-butyl 3-methyl (3R) -3-fluoropyrrolidine-1,3-dicarboxylate (# 166). To a solution containing # 164 (355mg, 1.50 mmol, 1 eq.) And di-tert-butyl carbonate (400mg, 1.8 mmol, 1.2 eq.) In methanol (15.5 mL) was added 10% Pd / C (70 mg). The reaction was hydrogenated at 45 psi on a parr shaker for 22 hours, filtered over celite, and the filtrate concentrated in vacuo and purified by silica gel chromatography (Gradient: 0 to 30% ethyl acetate in heptane) to yield # 166 like a clear oil. (272 mg, 74%). 1H NMR (400 MHz, CDCl3), δ
[01002] [01002] Step 2B. Synthesis of 1-tert-butyl 3-methyl (3S) -3-fluoropyrrolidine-1,3-dicarboxylate (# 167). Compound # 165 (362mg, 1.53 mmol, 1 eq.) Was converted to # 167 in 63% yield using the method described above for # 164. 1H NMR (400 MHz, CDCl3), δ 3.87 (s, 3H), 3.85-3.66 (m, 3H), 3.56 (m, 1H), 2.53-2.28 (m, 2H), 1.51 (s, 9H).
[01003] [01003] Step 3A. Synthesis of (3R) -1- (tert-butoxycarbonyl) -3-fluoropyrrolidine-3-carboxylic acid (# 168). To a solution of # 166 (272mg, 1.10 mmol, 1 eq.) Dissolved in methanol (2.96 mL) was added an aqueous sodium hydroxide solution (2.5 M,
[01004] [01004] Step 3B. Synthesis of (3S) -1- (tert-butoxycarbonyl) -3-fluoropyrrolidine-3-carboxylic acid (# 169). To a solution of # 167 (238 mg, 0.963 mmol, 1 eq.) Dissolved in methanol (2.6 ml) was added an aqueous sodium hydroxide solution (2.5 M, 0.88 ml) and the reaction was stirred at room temperature for 3 hours. The reaction was then quenched with 10% aqueous citric acid (5 ml) and ethyl acetate (100 ml) was added, and the layers were separated. The organic layer was washed with 10% citric acid, water, and brine, then dried over sodium sulfate, filtered and concentrated in vacuo to yield # 169 as a white solid (221 mg, 99%). 1H NMR (400 MHz, CDCl3), δ 3.96-3.69 (m, 3H), 3.59 (m, 1H), 2.59-2.33 (m, 2H), 1.51 (s, 9H). LC-MS (Protocol Q1): m / z 232.1 [M-H +], retention time = 0.67 minutes. Chiral HPLC retention time: 3.95 min (purity = 98%) (Column: Chiralpak AD-H, 4.6mm x 25cm, mobile phase 5-60% CO2 / Methanol, Flow Rate 3.0 mL / min); Optical rotation: [] D25 -3.6 (c = 0.55, MeOH)
[01005] [01005] Step 4A. Synthesis of lithium (3R) -1- (tert-butoxycarbonyl) -3-fluoropyrrolidine-3-carboxylate (# 170). To a solution of # 168 (50 mg, 0.21 mmol, 1 eq.) In methanol (0.2 ml) was added a solution of lithium hydroxide (9.2 mg, 0.38 mmol, 1.8 eq.) Dissolved in water (0.1 ml). Then, tetrahydrofuran (0.3 mL) was added and the reaction was stirred at 45 ° C for 18 hours. The reaction was concentrated in vacuo and the material was azeotroped (3X) with toluene (2 ml) to obtain # 170 (51mg, 100%) as a white solid which was used in the next step without further purification.
[01006] [01006] Step 4B. Synthesis of lithium (3S) -1- (tert-butoxycarbonyl) -3-fluoropyrrolidine-3-carboxylate (# 171). To a solution of # 169 (75 mg, 0.32 mmol, 1 eq.) In methanol (0.3 ml) was added a solution of lithium hydroxide (13.8 mg, 0.572 mmol, 1.8 eq.) In water (0.4 ml). Then, tetrahydrofuran (0.45 mL) was added and the reaction was stirred at 45 ° C for 18 hours. The reaction was concentrated in vacuo and the material was azeotroped (3X) with toluene (4 ml) to obtain # 171 (77 mg, 100%) as a white solid, which was used in the next step without further purification.
[01007] [01007] Step 5A. Synthesis of N - {(2R, 3R) -3 - [(2S) -1 - {(3R, 4S, 5S) -4 - [(N - {[(3R) -3-fluoropyrrolidin-3-yl] carbonyl } -L-valyl) (methyl) amino] -3-methoxy-5-methylheptanoyl} pyrrolidin-2-yl] -3-methoxy-2-methylpropanoyl} -L-methyl-phenylalaninate, trifluoroacetic acid salt (# 172) . At a suspension of # 168 (36.9 mg, 0.158 mmol, 1 eq.) And # 114 (100 mg,
[01008] [01008] Step 5B. Synthesis of N - {(2R, 3R) -3 - [(2S) -1 - {(3R, 4S, 5S) -4 - [(N - {[(3R) -3-fluoropyrrolidin-3-yl] carbonyl } -L-valyl) (methyl) amino] -3-methoxy-5-methylheptanoyl} pyrrolidin-2-yl] -3-methoxy-2-methylpropanoyl} -L-methyl-phenylalaninate, trifluoroacetic acid salt (# 173) . At a suspension of # 169 (36.9 mg, 0.158 mmol, 1eq.) And # 114 (100.0 mg,
[01009] [01009] Preparation of (2S) -N - [(2S) -1 - {[(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[2- (cyclohepta-2,4,6-trien-1-yl) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan -4-yl] (methyl) amino} -3-methyl-1-oxobutan-2-yl] -2-
[01010] [01010] Step 1. Synthesis of (3R, 4S, 5S) -4 - [{N - [(9H-fluoren-9-ylmethoxy) carbonyl] -L-valyl} (methyl) amino] -3-methoxy-5 -pentafluorophenyl methylheptanoate (# 174). To a solution of # @ 5 (19.43 g, 37.03 mmol, 1 eq.) In dichloromethane (100 mL) and pyridine (5.86 g, 74.1 mmol, 2 eq.) Was added pentafluorophenyl trifluoroacetate (20.7 g,
[01011] [01011] Step 2. Synthesis of N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[2- (cyclohepta- 2,4,6- trien-1-yl) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N ~ 2 ~ - [(9H-fluoren-9-ylmethoxy) carbonyl] -N-methyl-L-valinamide (# 175).
[01012] [01012] Step 3. Synthesis of N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[2- (cyclohepta- 2,4,6- trien-1-yl) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl -L-valinamide (# 176). According to general procedure A, of # 175 (560 mg, 0.690 mmol, 1 eq.) In dichloromethane (9 mL), and N, N-diethylmine (6.0 mL), the desired crude compound was synthesized, which was purified by silica gel chromatography (Gradient: 0 to 50% methanol in dichloromethane) to yield # 176 (351 mg, 87%) as a yellow oil. LC-MS (Protocol Q1): m / z
[01013] [01013] Step 4A. Synthesis of (2S) -2 - {[(2S) -1 - {[(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[2- (cyclohepta -2,4,6-trien-1-yl) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4 tert-butyl -yl] (methyl) amino} -3-methyl-1-oxobutan-2-yl] carbamoyl} -2-methylpiperidine-1-carboxylate (# 177). According to general procedure D, # 176 (100 mg, 0.170 mmol, 1 eq.), # 156 (53.8 mg, 0.221 mmol, 1.3 eq.), Dichloromethane (4.5 mL), HATU (84.9 mg, 0.221 mmol , 1.3 eq.) And N, N-diisopropylethylmine (0.123 mL, 0.697 mmol, 4.1 eq.), The desired crude material was synthesized, which was purified by silica gel chromatography (Gradient: 0 to 100% ethyl acetate in heptane) to yield # 177 (145 mg, assume quantitative yield) as a white solid. LC-MS (Protocol Q1): m / z 814.7 [M + H +], retention time = 1.14 minutes.
[01014] [01014] Step 4B. Synthesis of (2R) -2 - {[(2S) -1 - {[(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[2- (cyclohepta -2,4,6-trien-1-yl) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4 tert-butyl -yl] (methyl) amino} -3-methyl-1-oxobutan-2-yl] carbamoyl} -2-methylpiperidine-1-carboxylate (# 179). According to general procedure D, # 176 (100 mg, 0.170 mmol, 1 eq.), # 157 (53.8 mg, 0.221 mmol, 1.3 eq.), Dichloromethane (4.5 mL), HATU (84.9 mg, 0.221 mmol , 1.3 eq.) And N, N-diisopropylethylmine (0.123 mL, 0.697 mmol, 4.1 eq.), The desired crude material was synthesized, which was purified by silica gel chromatography (Gradient: 0 to 100% ethyl acetate in heptane) to yield # 179 (155 mg, assume quantitative yield) as a white solid. LC-MS (Protocol Q1): m / z 814.7 [M + H +], retention time = 1.14 minutes.
[01015] [01015] Step 5A. Synthesis of (2S) -N - [(2S) -1 - {[(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[2- (cyclohepta- 2,4,6-trien-1-yl) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4- yl] (methyl) amino} -3-methyl-1-oxobutan-2-yl] -2-methylpiperidine-2-carboxamide, hydrochloride salt (# 178). According to general procedure C, # 177 (143 mg, 0.176 mmol, 1 eq.) And 4M hydrochloric acid solution in dioxane (2.0 mL) the desired material was synthesized as a gum (145 mg). A portion of this crude residue (25 mg) was azeotroped with a methanol / acetonitrile mixture to yield # 178 (20 mg, 89% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6), δ 8.96-9.07 (m), 8.79-8.96 (m),
[01016] [01016] Step 5B. Synthesis of (2R) -N - [(2S) -1 - {[(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[2- (cyclohepta- 2,4,6-trien-1-yl) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4- yl] (methyl) amino} -3-methyl-1-oxobutan-2-yl] -2-methylpiperidine-2-carboxamide, hydrochloride salt (# 180). According to general procedure C, # 179 (162 mg, 0.199 mmol, 1 eq.) And 4M hydrochloric acid solution in dioxane (2.0 mL) the desired material was synthesized as a gum (155 mg). A portion of this gum (25 mg) was azeotroped with 1/1 methanol / acetonitrile mixture to yield # 180 (20 mg, 83%) as a solid. 1H NMR (400 MHz, DMSO-d6), δ 9.02-9.13 (m), 8.83-8.93 (m), 8.39-8.46 (m), 8.00-8.06 (m), 7.78 (t), 7.24-7.30 (m ), 7.16-7.21 (m), 6.54-6.65 (m), 6.09-6.19 (m), 5.11-5.18 (m), 4.69-4.78 (m), 4.59-4.68 (m), 4.46-4.56 (m) , 4.08-4.13 (m), 3.95-4.03 (m), 3.77-
[01017] [01017] Preparation of 2-methyl-L-prolyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[2- (cyclohepta-2 , 4,6-trien-1-yl) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl ] -N-methyl-L-valinamide, formic acid salt (# 182) and N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[ 2- (cyclohepta-2,4,6-trien-1-yl) ethyl] amino} -1- methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1 -oxoheptan-4-yl] -N ~ 2 ~ - {[(3R) -3-fluoropyrrolidin-3-yl] carbonyl} -N-methyl-L-valinamide, trifluoroacetic acid salt (# 184) and N- [ (3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[2- (cyclohepta-2,4,6-trien-1-yl) ethyl] amino} - 1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N ~ 2 ~ - {[(3S) -3-fluoropyrrolidin -3-yl] carbonyl} -N-methyl-L-valinamide, trifluoroacetic acid salt (# 186).
[01018] [01018] Step 1A. Synthesis of 1- (tert-butoxycarbonyl) -2-methyl-L-prolyl-N - [(3R, 4S, 5S) -1- {(2S) -2 - [(1R, 2R) -3 - {[2 - (cyclohepta-2,4,6-trien-1-yl) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1- oxoheptan-4-yl] -N-methyl-L-valinamide (# 181). According to general procedure D, of # 176 (100 mg, 0.170 mmol, 1 eq.), (S) -1- (tert-butoxycarbonyl) -2-methyl-L-proline (50.7 mg, 0.221 mmol, 1.3 eq.), dichloromethane (4.3 ml), HATU (84.9 mg, 0.221 mmol, 1.3 eq.) and N, N-diisopropylethylmine (0.123 ml, 0.697 mmol, 4.1 eq.), the desired crude material was synthesized, which was purified by silica gel chromatography (Gradient: 0 to 100% ethyl acetate in heptane) to yield # 181 (142 mg, assuming quantitative yield). LC-MS (Protocol Q1): m / z 800.6 [M + H +], retention time = 1.11 minutes.
[01019] [01019] Step 1B. Synthesis of (3R) -3 - {[(2S) -1 - {[(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[2- (cyclohept -2,4,6-trien-1-yl) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4 tert-butyl -yl] (methyl) amino} -3-methyl-1-oxobutan-2-yl] carbamoyl} -3-fluoropyrrolidine-1-carboxylate (# 183). To a solution of # 170 (18.2 mg,
[01020] [01020] Step 1C. Synthesis of (3S) -3 - {[(2S) -1 - {[(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[2- (cyclohepta -2,4,6-trien-1-yl) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4 -yl] (methyl) amino} -3-methyl-1-oxobutan-2-yl] carbamoyl} -3- tert-butyl fluorocyclopentanecarboxylate (# 185). To a solution of # 171 (24 mg,
[01021] [01021] Step 2A. Synthesis of 2-methyl-L-prolyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[2- (cyclohepta-2,4, 6-trien-1-yl) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-valinamide, formic acid salt (# 182).
[01022] [01022] Step 2B. Synthesis of N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[2- (cyclohepta- 2,4,6-trien-1-yl) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl- 1-oxoheptan-4-yl] -N ~ 2 ~ - {[(3R ) -3-fluoropyrrolidin-3-yl] carbonyl} -N-methyl-L-valinamide trifluoroacetic acid salt (# 184). According to general procedure C, from # 183 (61.1 mg, 0.076 mmol, 1 eq.), Dichloromethane (0.3 mL), and a 4M solution of hydrochloric acid in dioxane (0.9 mL) the desired crude material was synthesized ( 140 mg).
[01023] [01023] Step 2C. Synthesis of N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[2- (cyclohepta- 2,4,6-trien-1-yl) ethyl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl- 1-oxoheptan-4-yl] -N ~ 2 ~ - {[(3S ) -3-fluoropyrrolidin-3-yl] carbonyl} -N-methyl-L-valinamide, trifluoroacetic acid salt (# 186). According to general procedure C, of # 185
[01024] [01024] Preparation of (2S) -N - [(2S) -1 - {[(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1- methoxy-2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} - 5- methyl-1-oxoheptan-4-yl] (methyl) amino} -3-methyl-1-oxobutan-2-yl] -2-methylpiperidine-2-carboxamide, formate salt (# 188) and (2R) -N - [(2S) -1 - {[(3R, 4S, 5S) -3-methoxy-1 - {(2S) - 2 - [(1R, 2R) -1-methoxy-2-methyl-3-oxo- 3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] (methyl) amino} -3-methyl-1-oxobutan-2-yl] -2-methylpiperidine-2-carboxamide, formate salt (# 190) and N ~ 2 ~ - {[(3R) - 3-fluoropyrrolidin- 3-yl] carbonyl} -N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3-oxo- 3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide, trifluoroacetic acid salt. (# 192) and N ~ 2 ~ - {[(3S) -3-fluoropyrrolidin-3-yl] carbonyl} -N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1- methoxy-2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl ] pyrrolidin-1-yl} - 5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide, salt of trifluoroacetic acid. (# 194).
[01025] [01025] Step 1A. Synthesis of (2S) -2 - {[(2S) -1 - {[(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2- [(1R, 2R) -1-methoxy- 2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} -5-methyl- Tert-butyl 1-oxoheptan-4-yl] (methyl) amino} -3-methyl-1-oxobutan-2-yl] carbamoyl} -2-methylpiperidine-1-carboxylate (# 187). According to general procedure D, # 86 (280 mg, 0.4 mmol, 1 eq.), # 156 (100 mg, 0.4 mmol, 1 eq.), Dichloromethane (5mL), HATU (182 mg, 0.48 mmol, 1.2 eq.) And N, N-diisopropylethylmine (100 mg, 0.8 mmol, 2 eq.) The desired crude material was synthesized, which was purified by silica gel chromatography (Gradient: 0.01 to 0.05% methanol in dichloromethane) to render # 187 (220 mg, 62%) as a white solid. HPLC (Protocol V): m / z 883.57 [M + H +], retention time = 3.23 minutes (purity = 95%).
[01026] [01026] Step 1B. Synthesis of (2R) -2 - {[(2S) -1 - {[(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2- [(1R, 2R) -1-methoxy- 2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} -5-methyl- Tert-butyl 1-oxoheptan-4-yl] (methyl) amino} -3-methyl-1-oxobutan-2-yl] carbamoyl} -2-methylpiperidine-1-carboxylate (# 189). According to general procedure D, # 86 (400 mg, 0.6 mmol, 1 eq.), # 157 (146 mg, 0.6 mmol, 1 eq.), Dichloromethane (10 mL), HATU (259 mg, 0.72 mmol , 1.2 eq.) And N, N-diisopropylethylmine (158 mg, 1.2 mmol, 2 eq.) The desired crude material was synthesized, which was purified by silica gel chromatography (Gradient: 0.01 to 0.05% methanol in dichloromethane) to yield # 189 (220 mg, 37%) as a white solid.
[01027] [01027] Step 1C. Synthesis of (3R) -3-fluoro-3 - {[(2S) -1 - {[(3R, 4S, 5S) -3-methoxy-1 - {(2S) - 2 - [(1R, 2R) - 1-methoxy-2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} - Tert-butyl 5-methyl-1-oxoheptan-4-yl] (methyl) amino} -3-methyl-1-oxobutan-2-yl] carbamoyl} pyrrolidine-1-carboxylate (# 191). According to general procedure D, # 86 (300 mg, 0.45 mmol, 1 eq.), # 168 (106 mg, 0.45 mmol, 1 eq.), Dichloromethane (10 mL), HATU (194 mg, 0.54 mmol) , 1.2 eq.) And diisopropylethylmine (117 mg, 0.9 mmol, 2 eq.) The desired crude material was synthesized, which was purified by reverse phase chromatography (Method P) to yield # 191 (159 mg, 40%) as a white solid. HPLC (Protocol X): m / z 873.4 [M + H +], retention time = 3.32 minutes (purity = 99%).
[01028] [01028] Step 1D. Synthesis of (3S) -3-fluoro-3 - {[(2S) -1 - {[(3R, 4S, 5S) -3-methoxy-1 - {(2S) - 2 - [(1R, 2R) - 1-methoxy-2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} - Tert-butyl 5-methyl-1-oxoheptan-4-yl] (methyl) amino} -3-methyl-1-oxobutan-2-yl] carbamoyl} pyrrolidine-1-carboxylate (# 193). According to general procedure D, # 86 (300 mg, 0.45 mmol, 1 eq.), # 169 (106 mg, 0.45 mmol, 1 eq.), Dichloromethane (10 mL), HATU (194 mg, 0.54 mmol) , 1.2 eq.) And diisopropylethylmine (117 mg, 0.9 mmol, 2 eq.) The desired crude material was synthesized, which was purified by reverse phase chromatography (Method P) to yield # 193 (149 mg, 37%) as a white solid. HPLC (Protocol X): m / z 873.4 [M + H +], retention time = 3.34 minutes (purity = 98%).
[01029] [01029] Step 2A. Synthesis of (2S) -N - [(2S) -1 - {[(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2- [(1R, 2R) -1-methoxy-2 -methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} -5-methyl-1 -oxoheptan-4-yl] (methyl) amino} -3-methyl-1-oxobutan-2-yl] -2-methylpiperidine-2-carboxamide, formic acid salt (# 188). According to general procedure C, of # 187 (20 mg, 0.023 mmol, 1 eq.),
[01030] [01030] Step 2B. Synthesis of (2R) -N - [(2S) -1 - {[(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2- [(1R, 2R) -1-methoxy-2 -methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin-1-yl} -5-methyl-1 -oxoheptan-4-yl] (methyl) amino} -3-methyl-1-oxobutan-2-yl] -2-methylpiperidine-2-carboxamide, formic acid salt. (# 190). According to general procedure C, # 189 (20 mg, 0.022 mmol, 1 eq.), Dichloromethane (0.1 mL), acetonitrile (0.1 mL) and 4M hydrochloric acid in dioxane solution (0.26 mL) were synthesized. crude desired material, which was purified by reverse phase chromatography (Method N *) to obtain # 190 (13.2 mg, 73%,) HPLC (Protocol T): m / z 783.7 [M + H +], retention time = 2.5 minutes (purity = 100%). 1 H NMR (400 MHz, DMSO-d6), δ 8.83-8.86 (m), 8.60-8.62 (m), 8.24-8.27 (m), 7.83-
[01031] [01031] Step 2C. Synthesis of N ~ 2 ~ - {[(3R) -3-fluoropyrrolidin-3-yl] carbonyl} -N- [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [( 1R, 2R) -1-methoxy-2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin- 1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide, salt of trifluoroacetic acid. (# 192). According to general procedure C, from # 191 (10 mg, 0.011 mmol, 1 eq.), Dichloromethane (0.1 mL), acetonitrile (0.1 mL) and 4M hydrochloric acid in dioxane solution (0.13 mL) was synthesized crude desired material, which was purified by reverse phase chromatography (Method M *) to obtain # 192 (5.1mg, 60%) HPLC (Protocol T): m / z 773.5 [M + H +], retention time = 2.43 minutes (purity = 100%). 1H NMR (400 MHz, DMSO-d6) 29 2 .32-9.44 (m), 9.17-9.21 (m), 8.91 (d), 8.63-8.69 (m), 8.38-8.43 (m), 8.22-8.27 (m),
[01032] [01032] Step 2D. Synthesis of N ~ 2 ~ - {[(3S) -3-fluoropyrrolidin-3-yl] carbonyl} -N- [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [( 1R, 2R) -1-methoxy-2-methyl-3-oxo-3 - {[(1S) -2-phenyl-1- (1,3-thiazol-2-yl) ethyl] amino} propyl] pyrrolidin- 1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide, salt of trifluoroacetic acid. (# 194). According to general procedure C, # 193 (10 mg, 0.011 mmol, 1 eq.), Dichloromethane (0.1 mL), acetonitrile (0.1 mL) and 4M hydrochloric acid in dioxane solution (0.13 mL) were synthesized crude desired material, which was purified by reverse phase chromatography (Method M *) to obtain # 194 (9 mg, 93%) HPLC (Protocol T): m / z 773.8 [M + H +], retention time = 2.42 minutes (purity = 100%). 1H NMR (400 MHz, DMSO-d6)  9.39-9.52 (m), 9.21-9.35 (m), 8.90 (d), 8.63-8.69 (m), 8.42-8.48 (m), 8.29 -8.34 (m) , 7.80 (dd), 7.66 (dd), 7.22-7.33 (m), 7.13-7.21 (m), 5.47-5.57 (m), 5.36-5.44 (m),
[01033] [01033] Preparation of 1,2-dimethyl-D-prolyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3- {[(2S) - 3- (4-aminophenyl) -1-methoxy-1-oxopropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl- 1-oxoheptan-4-yl] -N-methyl-L-valinamide, formate salt (# 200) and 1,2-dimethyl-D-prolyl-N - [(3R, 4S, 5S) -1 - {( 2S) -2 - [(1R, 2R) -3 - {[(2S) -3- (4-aminophenyl) -1-methoxy-1-oxopropan-2-yl] amino} -1-methoxy-2-methyl -3- oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide, formate salt (# 201).
[01034] [01034] Step 1. Synthesis of 1,2-dimethyl-D-prolyl-N - [(3R, 4S, 5S) -1-tert-butoxy-3-methoxy-5-methyl-1-oxoheptan-4-yl ] -N-methyl-L-valinamide (# 195). According to general procedure D, # 6 (7.75 g, 21.6 mmol, 1 eq.), # 152 (3.88 g, 21.6 mmol, 1 eq.), Dichloromethane (100 mL), HATU (9.8 g, 25.9 mmol , 1.2 eq.), And diisopropylethylmine (11.1 g, 86.4 mmol, 4 eq.) The desired crude material was synthesized, which was purified by silica gel chromatography (Gradient: 20 to 55% ethyl acetate in petroleum ether) to yield # 195 (11.1 g, quantitative yield) as a yellow oil.
[01035] [01035] Step 2. Synthesis of 1,2-dimethyl-D-prolyl-N - [(2R, 3S, 4S) -1-carboxy-2-methoxy-4-methylhexan-3-yl] -N-methyl- L-valinamide (# 196). In accordance with general procedure B, from # 195 (11.1 g, 21.6 mmol, 1 eq.), Dichloromethane (100 ml) and trifluoroacetic acid (40 ml) the desired crude material was synthesized to obtain # 196 (10.1 g, quantitative yield) that was used in the next step without further purification. LC-MS (Protocol Z): m / z 428.5 [M + H +], retention time = 0.9 minutes.
[01036] [01036] Step 3. Synthesis of 1,2-dimethyl-D-prolyl-N - [(3R, 4S, 5S) -3-methoxy-5-methyl-1-oxo-1- (pentafluorophenoxy) heptan-4- il] -N-methyl-L-valinamide (# 197). To a cold (0 ° C) solution of # 196 (4.0 g, 9.4 mmol, 1 eq.) In dichloromethane (40 ml) was added dropwise pyridine (2.95 g, 37.6 mmol, 4eq.) Followed by a solution of pentafluorophenyl trifluoroacetate (3.9 g, 13.6 mmol, 1.4 eq.) in dichloromethane (5 ml). The mixture was stirred at room temperature for one hour, and the solvent was concentrated in vacuo. The residue was purified by silica gel chromatography (Gradient: 1 to 10% methanol in dichloromethane) to yield compound # 197 (4.5 g, 81.2% (in three steps) as a white solid.
[01037] [01037] Step 4. Synthesis of 1,2-dimethyl-D-prolyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -2- carboxy-1 -methoxypropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide, salt of trifluoroacetic acid (# 198). To a cold (0 ° C) solution of # 197 (4.0 g, 7.4 mmol, 1 eq.) In dichloromethane (25 mL) was added dropwise diisopropylethylmine (3.4 g, 26.3 mmol, 3.5 eq.) Followed by a solution # 103 (2.3 g,
[01038] [01038] Step 5. Synthesis of 1,2-dimethyl-D-prolyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2- [(1R, 2R) -1 -methoxy-2-methyl-3-oxo-3- (pentafluorophenoxy) propyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 199). To a solution of # 198 (280 mg, 0.394 mmol, 1 eq.) In dichloromethane (2 ml) was added pyridine (75 mg, 0.94 mmol, 2.4 eq.) Followed by a solution of pentafluorophenyl trifluoroacetate (268 mg, 0.94 mmol, 2.4 eq.) in dichloromethane (1.5 mL). The mixture was stirred at room temperature for 2.5 hours, and the solvent was concentrated in vacuo. The residue was purified by silica gel chromatography (Gradient: 1 to 10% methanol in dichloromethane) to yield compound # 199 (279 mg, 39%) as a white solid. LC-MS (Protocol Q1): m / z 763.5 [M + H +], retention time = 0.93 minutes.
[01039] [01039] Step 6A. Synthesis of 1,2-dimethyl-D-prolyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2- [(1R, 2R) -1-methoxy-3- { [(2S) -1-methoxy-1-oxo-3- (1,2,3,4-tetrahydroquinolin-6-yl) propan-2-yl] amino} -2-methyl-3-oxopropyl] pyrrolidin-1 -il} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide, trifluoroacetic acid salt (# 200). To a mixture of # 199 (25 mg,
[01040] [01040] Step 6B. Synthesis of 1,2-dimethyl-D-prolyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) - 3 - {[(2S) -3- ( 4-aminophenyl) -1-methoxy-1-oxopropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan -4-yl] -N-methyl-L-valinamide, trifluoroacetic acid salt (# 201). To a mixture of # 199 (25 mg, 0.033 mmol, 1eq) and 4-amino-L-phenylalaninate (8.8 mg, 0.033 mmol, 1 eq.) In commercially available methyl dichloromethane (1.5 mL), N, N- diisopropylethylmine (30.2 mg, 2.31 mmol, 7 eq.) was added. The reaction was stirred for 5 minutes and N, N-dimethylformamide (0.5 ml) was added. After 4 hours, additional N, N-diisopropylethylmine (37.75 mg, 2.88 mmol, 8.25 eq.) Was added and the mixture stirred for 50 minutes. Additional N, N-dimethylformamide (0.75 mL) was added and the reaction was stirred for 66 hours, concentrated in vacuo and the crude product was purified by reverse phase chromatography (Method M *) to give # 201 (14.3 mg, 56% ); HPLC (Protocol T): m / z
[01041] [01041] Preparation of 1,2-dimethyl-L-prolyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2- [(1R, 2R) -1-methoxy- 3 - {[(2S) -1-methoxy-1-oxo-3- (1,2,3,4-tetrahydroquinolin-6-yl) propan-2-yl] amino} -2-methyl-3-oxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide, formate salt. (# 207) and 1,2-dimethyl-L-prolyl-N - [(3R, 4S, 5S) -1 - {(2S) -2- [(1R, 2R) -3 - {[(2S) - 3- (4-aminophenyl) -1-methoxy-1-oxopropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl- 1-oxoheptan-4-yl] -N-methyl-L-valinamide formate salt (# 208) and 1,2-dimethyl-L-prolyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2 - [(1R, 2R) -1- methoxy-3 - {[(2S) -1-methoxy-1-oxo-3-phenylpropan-2-yl] amino} -2-methyl- 3- oxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide, trifluoroacetic acid salt (# 209)
[01042] [01042] Step 1. Synthesis of 1,2-dimethyl-L-prolyl-N - [(3R, 4S, 5S) -1-tert-butoxy-3-methoxy-5-methyl-1-oxoheptan-4-yl ] -N-methyl-L-valinamide (# 202). According to general procedure D, # 6 (4.3 g, 12 mmol, 1 eq.), # 150 (2.15 g, 12 mmol, 1 eq.), Dichloromethane (50 mL), HATU (5.46 g, 14 mmol , 1.2 eq.), And diisopropylethylmine (8.17 mL) the desired crude material was synthesized, which was purified by silica gel chromatography (Gradient: 20 to 55% ethyl acetate in petroleum ether) to yield # 202 (5.2 g , 89%) as a yellow oil.
[01043] [01043] Step 2. Synthesis of 1,2-dimethyl-L-prolyl-N - [(2R, 3S, 4S) -1-carboxy-2-methoxy-4-methylhexan-3-yl] -N-methyl- L-valinamide (# 203). According to general procedure B, # 202 (5.2 g, 10.77 mmol, 1 eq.), Dichloromethane (45 ml), and trifluoroacetic acid (20 ml) were synthesized the desired crude material, to obtain # 203 (7 g , quantitative yield) that was used in the next step without further purification.
[01044] [01044] Step 3. Synthesis of 1,2-dimethyl-L-prolyl-N - [(3R, 4S, 5S) -3-methoxy-5-methyl-1-oxo-1- (pentafluorophenoxy) heptan-4- il] -N-methyl-L-valinamide (# 204). To a cold (0 ° C) solution of # 203 (7.0 g, 10.77 mmol, 1 eq.) In dichloromethane (15 mL) was added dropwise pyridine (3.41 g 43.08 mmol, 4eq.) Followed by a solution of trifluoroacetate of pentafluorophenyl (6.03 g, 21.54 mmol, 2 eq.) in dichloromethane (7 mL). The mixture was stirred at room temperature for one hour, and the solvent was concentrated in vacuo. The residue was purified by silica gel chromatography (Gradient: 1 to 10% methanol in dichloromethane) to yield compound # 204 (8 g, 82% in two steps) as a yellow solid.
[01045] [01045] Step 4. Synthesis of 1,2-dimethyl-L-prolyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -2- carboxy-1 -methoxypropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 205). To a cold (0 ° C) solution of # 204 (8.0 g, 10.77 mmol, 1 eq.) In dichloromethane (25 ml) was added dropwise diisopropylethylmine (5.6 g, 43.08 mmol, 4 eq.) Followed by a solution of # 103 (3.22 g, 10.77 mmol, 1 eq.) in dichloromethane (15 ml). After the addition, the mixture was stirred at room temperature for 16 hours and the solvent was removed in vacuo. The residue was purified by silica gel chromatography (Gradient: 1 to 10% methanol in dichloromethane) to give # 205 (2.2 g, 33%) as a yellow HPLC solid (Protocol X): m / z 597.42 [M + H + ], retention time = 8,729 minutes (purity> 97%), chiral HPLC retention time: 2.87 min (purity = 89%) Column: Chiralcel OD-3, 150 x 4.6 mm, 3 m; Mobile phase: ethanol (0.05% diethylmine) in CO2 from 5% to 40% over 12 minutes; Flow Rate: 2.5 mL / minute.
[01046] [01046] Step 5. Synthesis of 1,2-dimethyl-L-prolyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2- [(1R, 2R) -1 -methoxy-2-methyl-3-oxo-3- (pentafluorophenoxy) propyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (# 206). To a solution of # 198 (0.28 g, 0.47 mmol, 1 eq.) In dichloromethane (2 ml) was added pyridine (75 mg, 0.94 mmol, 2 eq.) Followed by a solution of pentafluorophenyl trifluoroacetate (268 mg, 0.94 mmol, 2 eq.) in dichloromethane (1.5 mL). The mixture was stirred at room temperature for 2.5 hours, and the solvent was concentrated in vacuo. The residue was purified by silica gel chromatography (Gradient: 1 to 10% methanol in dichloromethane) to yield compound # 206 (348 mg, 97%) as a white solid. LC-MS (Protocol Q1): m / z 763.5 [M + H +], retention time = 0.9 minutes.
[01047] [01047] Step 6A. Synthesis of 1,2-dimethyl-L-prolyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2- [(1R, 2R) -1-methoxy-3- { [(2S) -1-methoxy-1-oxo-3- (1,2,3,4-tetrahydroquinolin-6-yl) propan-2-yl] amino} -2-methyl-3-oxopropyl] pyrrolidin-1 -yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide, salt of trifluoroacetic acid. (# 207). The title compound was prepared from # 206 (25 mg, 0.033 mmol, 1 eq.) And # 215 (7.7 mg, 0.033 mmol, 1 eq) using the method described above for preparing # 200. The crude product was purified by reverse phase chromatography (Method M *) to give # 207 (11.7 mg, 44%). HPLC (Protocol T): m / z 407.6, double charge [2+], retention time = 1.59 minutes (purity = 100%). 1H NMR (DMSO-d6) 29.57-9.69 (m), 28.68-8.76 (m), 8.42-8.47 (m),
[01048] [01048] Step 6B. Synthesis of 1,2-dimethyl-L-prolyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3- {[(2S) -3- ( 4-aminophenyl) -1-methoxy-1-oxopropan-2-yl] amino} -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan -4-yl] -N-methyl-L-valinamide, trifluoroacetic acid salt (# 208). To a mixture of # 206 (25.0 mg, 0.033 mmol, 1 eq.), And the commercially available methyl 4-amino-L-phenylalaninate (8.8 mg, 0.033 mmol, 1 eq.) In dichloromethane (1.5 mL), N , N-diisopropylethylmine (30.2 mg, 2.31 mmol, 7 eq.) Was added. The reaction was stirred for 5 minutes and N, N-dimethylformamide (0.5 ml) was added. After 2 ½ hours, additional N, N-diisopropylethylmine (30.2 mg, 2.31 mmol, 7 eq.) Was added. After stirring for 3 1/2 hours, additional N, N-dimethylformamide (0.75 mL) was added and The reaction was stirred for 66 hours, concentrated in vacuo and the crude product was purified by reverse phase chromatography (Method
[01049] [01049] Step 6C. Synthesis of 1,2-dimethyl-L-prolyl-N - [(3R, 4S, 5S) -3-methoxy-1 - {(2S) -2- [(1R, 2R) -1-methoxy-3- { [(2S) -1-methoxy-1-oxo-3-phenylpropan-2-yl] amino} -2-methyl-3-oxopropyl] pyrrolidin-1-yl} -5-methyl-1-oxoheptan-4-yl ] -N-methyl-L-valinamide, trifluoroacetic acid salt (# 209). The title compound was prepared from # 206 (25.0 mg, 0.033 mmol, 1 eq.) And methyl-L-phenylalaninate hydrochloride (7.1 mg, 0.033 mmol, 1 eq.) Using the method described above for preparing # 200. The crude product was purified by reverse phase chromatography (Method M *) to give # 209 (10.3 mg, 41%). HPLC (Protocol T): m / z 758.7 [M + H +], retention time = 1,787 minutes (purity = 100%). 1H NMR (DMSO-d6) 29.57-9.70 (m), 8.70-8.75 (m) , 8.50-8.56 (m), 8.32-8.42 (m), 8.24-8.26 (m), 8.10-8.15 (m), 7.14-7.27 (m), 7.12-7.13 (m), 6.98-7.00 (m), 4.69-
[01050] [01050] Preparation of methyl (2S) -2-amino-3- (1,2,3,4-tetrahydroquinolin-6-yl) propanoate
[01051] [01051] Step 1. Synthesis of 6- (bromomethyl) quinoline (# 210). A solution of 6-methylquinoline (5 g, 35 mmol, 1 eq.), N-Bromosuccinimide (8.1 g, 45.5 mmol, 1.3 eq.) And benzyl peroxide (840 mg, 3.5 mmol, 0.1 eq.) In tetrachloride carbon (100 mL) was stirred at reflux for 3 hours and then cooled to room temperature. The reaction mixture was filtered and the filtrate was concentrated in vacuo. The residue was dissolved in tetrahydrofuran (100 ml) and filtered. The filtrate was used directly in the next step without further purification
[01052] [01052] Step 2. Synthesis of 6 - {[(2S, 5R) -3,6-dimethoxy-5- (propan-2-yl) -2,5-dihydropyrazin-2-yl] methyl} quinoline (# 211 ). To a cold (-70 ° C) solution of (2R) -3,6-dimethoxy-2- (propan-2-yl) -2,5-dihydropyrazine (25.8 g, 140 mmol, 2 eq.) In tetrahydrofuran ( 200 ml) was added dropwise n-butyllithium (2.5 M, 64.4 ml, 161 mmol. 2.3eq.) And then stirred for 30 minutes. A solution of # 210 (15.4 g, 70 mmol, 1 eq.) In tetrahydrofuran (150 ml) was added dropwise at -65 ° C and then the solution was stirred for 2 hours at this temperature. The reaction was cooled by saturated aqueous ammonium chloride (100 ml) and extracted with ethyl acetate (100 ml). The organic phase was dried over sodium sulfate and concentrated in vacuo. The residue was purified by silica column chromatography (Gradient: 10 to
[01053] [01053] Step 3. Synthesis of methyl (2S) -2-amino-3- (quinolin-6-yl) propanoate (# 212).
[01054] [01054] Step 4. Synthesis of methyl (2S) -2 - [(tert-butoxycarbonyl) amino] -3- (quinolin-6-yl) propanoate (# 213). To a solution # 212 (5.2 g, 22.5 mmol, 1 eq.) And triethylmine (9.1 g, 90 mmol, 4eq.) In mixed solvent of methanol (30 mL) and water (50 mL) was added di-tert-dicarbonate -butyl (17.5 g, 78.75 mmol, 3.5 eq.) at 0 ° C and then the solution was stirred at 10 ° C overnight. The reaction mixture was filtered and the filter cake was washed with methanol (20 ml X 2). The filtrate was extracted with ethyl acetate (50 ml X 2) and the organic phase was concentrated in vacuo. The residue was purified by silica column chromatography (Gradient: 25 to 50% ethyl acetate in petroleum ether) to yield # 213 (5.5 g, 74% in two steps) as a yellow oil. LC-MS (Protocol Z): m / z 331.2 [M + H +], retention time =
[01055] [01055] Step 5. Synthesis of methyl (2S) -2 - [(tert-butoxycarbonyl) amino] -3- (1,2,3,4-tetrahydroquinolin-6-yl) propanoate (# 214) A suspension of # 213 (1.5 g,
[01056] [01056] Step 6. Synthesis of methyl (2S) -2-amino-3- (1,2,3,4-tetrahydroquinolin-6-yl) propanoate (# 215). To a solution of # 214 (750 mg, 2.25 mmol, 1 eq.) In dichloromethane (20 ml) was added dropwise trifluoroacetic acid (2 ml) at 0 ° C and then the solution was stirred at 20 ° C for in the evening. The reaction mixture was concentrated in vacuo and the residue was dissolved in water (20 ml). The solution was basified with sodium carbonate and extracted with ethyl acetate / tetrahydrofuran (30 mL X 3). The organic phase was dried over sodium sulfate and concentrated in vacuo to yield # 215 (450 mg, 85%) as a yellow oil. 1H NMR (400 MHz, CDCl3): δ 6.70 (d, 2H), 6.40 (m, 1H), 3.73 (s, 3H), 3.67 (m, 1H), 3.30 (m, 2H), 2.96 (m, 1H ),
[01057] [01057] Preparation of N - {(2R, 3R) -3 - [(2S) -1 - {(3R, 4S, 5S) -4 - [(N - {[(3R) -3- fluoropyrrolidin-3- yl] carbonyl} -L-valyl) (methyl) amino] -3-methoxy-5-methylheptanoyl} pyrrolidin-2-yl] -3-methoxy-2-methylpropanoyl} -L-phenylalanine, trifluoroacetic acid salt (# 217 ) and N - {(2R, 3R) -3 - [(2S) -1 - {(3R, 4S, 5S) -4 - [(N - {[(3S) -3-fluoropyrrolidin-3-yl] carbonyl } -L-valyl) (methyl) amino] -3-methoxy-5-methylheptanoyl} pyrrolidin-2-yl] -3-methoxy-2-methylpropanoyl} - L-phenylalanine, trifluoroacetic acid salt (# 219).
[01058] [01058] Step 1A. Synthesis of N - {(2R, 3R) -3 - [(2S) -1 - {(3R, 4S, 5S) -4 - [(N - {[(3R) -1- (tert-butoxycarbonyl) -3 -fluoropyrrolidin-3-yl] carbonyl} -L-valyl) (methyl) amino] -3-methoxy-5-methylheptanoyl} pyrrolidin-2-yl] -3-methyl-2-methylpropanoyl} -L-phenylalaninate ( # 216). To a solution of # 168 (36.9 mg, 0.158 mmol, 1 eq.) And # 114 (100 mg, 0.158 mmol, 1 eq.) In dichloromethane (3.6 mL) and N, N-dimethylformamide (0.8 mL), was added diisopropylethylmine (0.083 mL, 0.474 mmol, 3 eq.) followed by HATU (60.7 mg, 0.158 mmol, 1eq.). The reaction was allowed to stir at room temperature for 18 hours, diluted with ethyl acetate (25 mL), washed with water (1X), 10% citric acid (1X) and brine (1X). The organic layer was dried over sodium sulfate, filtered, and the filtrate concentrated in vacuo to give crude # 216 (220 mg, 164% theory) which was used in the next step without further purification. HPLC (Protocol Q): m / z 848.6 [M + H +], retention time = 2.10 minutes.
[01059] [01059] Step 1B. Synthesis of N - {(2R, 3R) -3 - [(2S) -1 - {(3R, 4S, 5S) -4 - [(N - {[(3S) -1- (tert-butoxycarbonyl) -3 -fluoropyrrolidin-3-yl] carbonyl} -L-valyl) (methyl) amino] -3-methoxy-5-methylheptanoyl} pyrrolidin-2-yl] -3-methyl-2-methylpropanoyl} -L-phenylalaninate ( # 218). To a solution of # 169 (36.9 mg, 0.158 mmol, 1 eq.) And # 114 (100 mg, 0.158 mmol, 1 eq.) In dichloromethane (3.6 mL) and N, N-dimethylformamide (0.8 mL), was added diisopropylethylmine (0.083 mL, 0.474 mmol, 3 eq.) followed by HATU (60.7 mg, 0.158 mmol, 1eq.). The reaction was allowed to stir at room temperature for 18 hours, diluted with ethyl acetate (25 mL), washed with water (1 X), 10% citric acid (1X) and brine (1X). The organic layer was dried over sodium sulfate,
[01060] [01060] Step 2A. Synthesis of N - {(2R, 3R) -3 - [(2S) -1 - {(3R, 4S, 5S) -4 - [(N - {[(3R) -3-fluoropyrrolidin-3-yl] carbonyl } -L-valyl) (methyl) amino] -3-methoxy-5-methylheptanoyl} pyrrolidin-2-yl] -3-methoxy-2-methylpropanoyl} -L-phenylalanine, trifluoroacetic acid salt (# 217). To a solution of crude # 216 (134 mg) in tetrahydrofuran (4 ml) was added lithium hydroxide (1M, 0.5 ml). The reaction was stirred at room temperature for 18 hours and concentrated in vacuo. The residue was dissolved in dichloromethane (2 ml) and trifluoroacetic acid (2 ml) was added. The reaction was stirred for 4 hours and concentrated in vacuo. The crude material was purified by reverse phase chromatography (Method M *) to obtain # 217 (60 mg, 86% in two steps) as a gum. LC-MS (Protocol Q): m / z 734.93 [M + H +], retention time = 1.19 minutes. 1 H NMR (DMSO-d6) 212.62-12.83 (m), 9.30-9.43 (m), 9.17-9.28 (m), 8.34-8.41 (m),
[01061] [01061] Step 2B. Synthesis of N - {(2R, 3R) -3 - [(2S) -1 - {(3R, 4S, 5S) -4 - [(N - {[(3S) -3-fluoropyrrolidin-3-yl] carbonyl } -L-valyl) (methyl) amino] -3-methoxy-5-methylheptanoyl} pyrrolidin-2-yl] -3-methoxy-2-methylpropanoyl} -L-phenylalanine, trifluoroacetic acid salt (# 219). To a solution of crude # 218 (100 mg) in tetrahydrofuran (4 ml) was added 1.0 M lithium hydroxide in water (0.5 ml). The reaction was stirred at room temperature for 18 hours and then concentrated in vacuo. The crude material was dissolved in dichloromethane (2 ml) and trifluoroacetic acid (2 ml) was added. The reaction was stirred for 4 hours and then concentrated in vacuo. The crude material was purified by reverse phase chromatography (Method M *) to obtain # 219 as a gum (60 mg, 91% in two steps). LC-MS (Protocol Q): m / z 734.97 [M + H +], retention time = 1.14 minutes. 1H NMR (400 MHz, DMSO-d6), δ 12.62-12.85 (m), 9.32-9.43 (m), 9.13-9.26 (m), 8.39-8.46 (m), 8.30-8.39 (m), 8.25-8.29 (m), 8.08-
[01062] [01062] Preparation of 2-methylillanyl-N - {(3R, 4S, 5S) -1 - [(2S) -2 - {(3R, 4R, 7S) -7-benzyl-4-methyl-18 - [( 4S, 5R) -5-methyl-2-oxoimidazolidin-4-yl] -5,8,13-trioxo-2-oxa-6,9,12-triazaoctadecan-3-yl} pyrrolidin-1-yl] -3 -methoxy-5-methyl-1-oxoheptan-4-yl} -N-methyl-L-valinamide (# 257).
[01063] [01063] Step 1. Synthesis of [(2S) -1 - ({2 - [(tert-butoxycarbonyl) amino] ethyl} amino) -1-oxo- 3-phenylpropan-2-yl] carbamate of 9H-fluoren 9-ylmethyl (# 253). Following general procedure D using N - [(9H-fluoren-9-ylmethoxy) carbonyl] -L-phenylalanine (500 mg, 1.29 mmol, 1.0 eq), tert-butyl (2-aminoethyl) carbamate (207 mg, 1.29 mmol , 1.0 eq.), HATU (620 mg, 1.55 mmol, 1.2 eq.) And Hunig's Base (0.452 mL, 2.58 mmol, 2.0 eq) in 6 mL of DMF # 253 was provided as a white solid (620 mg, 91 %) following solvent concentration and re-crystallization using ethyl acetate. LC-MS (Protocol Q1): m / z 552.3 [M + Na +], retention time = 1.01 minutes.
[01064] [01064] Step 2. Synthesis of N-alpha - [(9H-fluoren-9-ylmethoxy) carbonyl] -N- [2 - ({6- [(4S, 5R) -5-methyl-2-oxoimidazolidin-4 -yl] hexanoyl} amino) ethyl] -L-phenylalaninamide (# 254).
[01065] [01065] Step 3. Synthesis of N- [2 - ({6 - [(4S, 5R) -5-methyl-2-oxoimidazolidin-4-yl] hexanoyl} amino) ethyl] -L-phenylalaninamide (# 255) . Following general procedure A using # 254 (35 mg, 0.056 mmol, 1.0 eq.), Piperidine (0.10 mL, 1.0 mmol, 20 eq.) In 0.5 mL DMF followed by purification using silica chromatography (0-30% methanol in dichloromethane) provides # 253 (19 mg, 84%). LC-MS (Protocol Q1): m / z 404.2 [M + H +], retention time = 0.48 minutes.
[01066] [01066] Step 4. Synthesis of N - [(9H-fluoren-9-ylmethoxy) carbonyl] -2-methylillanyl-N- {(3R, 4S, 5S) -1 - [(2S) -2 - {(3R , 4R, 7S) -7-benzyl-4-methyl-18 - [(4S, 5R) -5-methyl-2-oxoimidazolidin-4-yl] -5,8,13-trioxo-2-oxa-6, 9,12-triazaoctadecan-3-yl} pyrrolidin-1-yl] -3-methoxy-5-methyl-1-oxoheptan-4-yl} -N-methyl-L-valinamide (# 256). Following general procedure D using # 105 (36.6 mg, 0.047 mmol, 1.0 eq.), # 255 (19 mg,
[01067] [01067] Step 5. Synthesis of 2-methylanil-N - {(3R, 4S, 5S) -1 - [(2S) -2 - {(3R, 4R, 7S) -7-benzyl-4-methyl-18 - [(4S, 5R) -5-methyl-2-oxoimidazolidin-4-yl] -5,8,13-trioxo-2-oxa-6,9,12- triazaoctadecan-3-yl} pyrrolidin-1-yl ] -3-methoxy-5-methyl-1-oxoheptan-4-yl} -N-methyl-L-valinamide (# 257). Following general procedure A using # 256 (5mg, 0.004 mmol, 1.0 eq.) And piperidine (0.02 mL, 0.2 mmol, 50 eq.) In 0.7 mL of DMF followed by purification (Method J) yielded # 257 (2 mg, 50%) as a colorless glass. LC-MS (Protocol Q1): m / z 1164.8 [M + H +], retention time = 0.99 minutes. 1H NMR (400
[01068] [01068] Preparation of N- [5- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) pentanoyl] -N, 2-dimethillanyl-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-carboxy-2-phenylethyl] amino} -1- methoxy-2-methyl-3-oxopropyl] pyrrolidin-1- il} -2-methoxy-1 - [(1S) -1-methylpropyl] -4-oxobutyl} -N-methyl-L-valinamide (mv # 115).
[01069] [01069] Step 1. Preparation of N- [5- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) pentanoyl] - N, 2-dimethylanil-N - {(1S, 2R ) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-carboxy-2-phenylethyl] amino} -1- methoxy-2-methyl-3-oxopropyl] pyrrolidin -1-yl} -2-methoxy-1 - [(1S) -1-methylpropyl] -4-oxobutyl} -N-methyl-L-valinamide (mv # 115). To a stirred solution of 5- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) pentanoic acid (12 mg, 0.061 mM) in 0.4 ml of dichloromethane, and
[01070] [01070] Preparation of N- [6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] -N, 2-dimethillanil-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-carboxy-2-phenylethyl] amino} -1- methoxy-2-methyl-3-oxopropyl] pyrrolidin-1- il} -2-methoxy-1 - [(1S) -1-methylpropyl] -4-oxobutyl} -N- methyl-L-valinamide (mc # 115)
[01071] [01071] Step 1. Synthesis of 6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl chloride (# 248). To a stirred solution of 6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoic acid (3.15 g, 14.9 mM) in 15 ml of dichloromethane, oxalyl chloride (1.61 ml, 17.9 mM) was added followed by one drop of DMF. The reaction was allowed to stir at room temperature for three hours. The reaction was concentrated in vacuo. The residue was dissolved in a one to one solution of heptane and dichloromethane and then concentrated in vacuo. This process was repeated two more times producing the solid
[01072] [01072] Step 2. Synthesis of N- [6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] -N, 2-dimethillanine (# 249). To a stirred solution of # 248 (600 mg, 2.61 mM) in 10 ml of dichloromethane, N, 2-dimethillanine (306 mg, 2.61 mM) was added followed by triethylmine (1.09 ml, 7.84 mM). The reaction was allowed to stir at room temperature for three hours. Dichloromethane was added to the reaction and the organic layer was washed three times with water and twice with brine. The organic layer was separated and then dried over sodium sulfate before being concentrated in vacuo. The crude residue was purified by chromatography on silica (0-30% methanol in dichloromethane) on silica that was previously neutralized with triethylmine yielding a # 249 white solid (127 mg, 16%). LC-MS (Protocol Q): m / z 309.0 [M-H-], retention time = 0.96 minutes.
[01073] [01073] Step 3. Synthesis of N - {(2R, 3R) -3-methoxy-3 - [(2S) -1 - {(3R, 4S, 5S) -3-methoxy-5-methyl-4- [ methyl (L-valyl) amino] heptanoyl} pyrrolidin-2-yl] -2-methylpropanoyl} -L-phenylalanine (# 250). To a stirred solution of # 113 (2.10 g, 2.46 mM) in 10 ml of THF, lithium hydroxide (228 mg, 5.16 mM) was added followed by 3 ml of water. The reaction was allowed to stir at room temperature for 2 hours. The reaction was acidified by adding 1 M HCl and then concentrated in vacuo. The resulting white solid was taken up in 20 ml of acetonitrile and 5 ml of water. The aqueous layer was removed and the organic layer was washed once with water. The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo.
[01074] [01074] Step 4. Synthesis of N- [6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] -N, 2-
[01075] [01075] Preparation of N- [4- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) butanoyl] -N, 2-dimethillanil-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-carboxy-2-phenylethyl] amino} -1- methoxy-2-methyl-3-oxopropyl] pyrrolidin-1- il} -2-methoxy-1 - [(1S) -1-methylpropyl] -4-oxobutyl} -N-methyl-L-valinamide (mb # 115).
[01076] [01076] Step 1. Synthesis of N- [4- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) butanoyl] -N, 2-dimethillanyl-N - {(1S, 2R ) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-carboxy-2-phenylethyl] amino} -1- methoxy-2-methyl-3-oxopropyl] pyrrolidin -1-yl} -2-methoxy-1 - [(1S) -1-methylpropyl] -4-oxobutyl} -N-methyl-L-valinamide (mb # 115). A stirred solution of 4- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) butanoic acid (1.2 equivalents), HATU (1.2 equivalents), and Hunig's Base (3 equivalents) in DMF and dichloromethane is allowed to stir for 30 minutes.
[01077] [01077] Preparation of N- [7- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) heptanoyl] -N, 2-dimethillanyl-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-carboxy-2-phenylethyl] amino} -1- methoxy-2-methyl-3-oxopropyl] pyrrolidin-1- il} -2-methoxy-1 - [(1S) -1-methylpropyl] -4-oxobutyl} -N-methyl-L-valinamide (me # 115).
[01078] [01078] Step 1. Synthesis of N- [7- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) heptanoyl] -N, 2-dimethillanyl-N - {(1S, 2R ) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-carboxy-2-phenylethyl] amino} -1- methoxy-2-methyl-3-oxopropyl] pyrrolidin -1-yl} -2-methoxy-1 - [(1S) -1-methylpropyl] -4-oxobutyl} -N-methyl-L-valinamide (me # 115). A stirred solution of 7- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) heptanoic acid (1.2 equivalents), HATU (1.2 equivalents), and Hunig's Base (3 equivalents) in DMF and dichloromethane is allowed to stir for 30 minutes.
[01079] [01079] Preparation of N- [6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] -L-valyl- N ~ 5 ~ -carbamoyl-N- (4- {(8S, 11S, 12R) -12- (2 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-carboxy-2-phenylethyl] amino} -1-methoxy -2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-oxoethyl) -8-isopropyl- 4,5,5,10-tetramethyl-11 - [(1S) -1-methylpropyl] -3,6 , 9-trioxo-2,13-dioxa-4,7,10-triazatetradec-1-yl} phenyl) -L-ornithinamide (mcValCitPABC # 115).
[01080] [01080] Step 1. Synthesis of N- [6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] -L-valyl-N ~ 5 ~ -carbamoyl-N- (4 - {(8S, 11S, 12R) -12- (2 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-carboxy-2-phenylethyl] amino} -} 1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -2-oxoethyl) -8-isopropyl- 4,5,5,10-tetramethyl-11 - [(1S) -1-methylpropyl] - 3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradec-1-yl} phenyl) -L-ornithinamide (mcValCitPABC # 115). A solution of mcCValCitPABC (Linker # D, 1 equivalent) and # 115 (1 equivalent) in DMF is prepared. Hunig's base (4 equivalents), 2,6-Luditine (4 equivalents), and HOAT (0.2 equivalents) is added. Reaction is monitored by LC-MS. Reaction is concentrated and purification is completed by pressure in Isco medium reverse phase chromatography (Gradient: 5% -100% water in acetonitrile).
[01081] [01081] Preparation of N- (21-amino-4,7,10,13,16,19-hexaoxahenicosan-1-oil) -N, 2-dimethillanil-N - {(1S, 2R) -4 - {( 2S) -2 - [(1R, 2R) -3 - {[(1S) -1-carboxy-2-phenylethyl] amino} -1- methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} - 2-methoxy-1 - [(1S) -1-methylpropyl] -4-oxobutyl} -N-methyl-L-valinamide (AmPeg6C2 # 115).
[01082] [01082] Step 1. Synthesis of N- (21-amino-4,7,10,13,16,19-hexaoxahenicosan-1-oil) - N, 2-dimethillanil-N - {(1S, 2R) -4 - {(2S) -2 - [(1R, 2R) -3 - {[(1S) -1-carboxy-2-phenylethyl] amino} -1- methoxy-2-methyl-3-oxopropyl] pyrrolidin-1- il} -2-methoxy-1 - [(1S) -1-methylpropyl] -4-oxobutyl} -N-
[01083] [01083] Preparation of 1,2-dimethyl-D-prolyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3- ({(2S) - 3- [4 - ({N- [6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] glycyl} amino) phenyl] -1- methoxy-1-oxopropan- 2-yl} amino) -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide mcGly # 201.
[01084] [01084] Step1: Synthesis of N- (tert-butoxycarbonyl) -4 - ({N- [6- (2,5-dioxo-2,5-dihydro-1H pyrrol-1-yl) hexanoyl] glycyl} amino) -L-methylphenylalaninate (# 251): To a solution of methyl 4-amino-N- (tert-butoxycarbonyl) -L-phenylalaninate (4.1 g, 15.3 mmol, 1eq.) In dry N, N-dimethylformamide (70 mL) N, N'-Dicyclohexylcarbodiimide (2.9 g, 15.3 mmol, 1eq.) was added at 0 ° C. The mixture was stirred at 0 oC for 30 minutes. A solution of 2- (6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanamido) acetic acid (3 g, 10.2 mmol, 0.66 eq.) In N, N-dimethylformamide (20 mL) was added at 0 ° C. The mixture was stirred at room temperature for 3 days. The mixture was filtered. The filtrate was poured into ice water (200 ml) and extracted with EtOAc (200 mlX3). The extract was washed with brine (200 ml), dried over Na2SO4 and concentrated in vacuo to yield # 251 (1.8 g, 32.3% yield) as a light yellow solid. HPLC (Protocol Q2) [M + Na +] 567.3, retention time = 1.02 min
[01085] [01085] Step 2: Synthesis of methyl 4 - ({N- [6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] glycyl} amino) -L-phenylalaninate (# 252): To a solution of # 251 (800 mg, 1.47 mmol, 1 eq.) In dichloromethane (16 mL) was added TFA (4.8 mL) at 0 oC.
[01086] [01086] Step 3: Synthesis of 1,2-dimethyl-D-prolyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3- ({( 2S) -3- [4 - ({N- [6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] glycyl} amino) phenyl] -1- methoxy-1 -oxopropan-2-yl} amino) -1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl- L-valinamide (mcGly # 201). To a solution of # 198 (94 mg, 0.13mmol, 1 eq.) And # 252 (60.3 mg, 0.18 mmol, 1.4 eq.) In N, N-dimethylformamide (2 mL) was added HATU (64.2 mg, 0.13 mmol , 1 eq.) Followed by N, N-diisopropylethylmine (66 mg, 0.52 mmol). The solution was stirred at room temperature for 1 hour. The reaction mixture was neutralized with aq. and concentrated to give crude product, which was purified by silica gel chromatography (eluted with 1% to 7% MeOH / DCM), then purified again by preparative TLC (Methanol: dichloromethane: = 1: 10) to give mcGly # 201 (25 mg, 16.2%) as a white solid: ESI-MS: m / z 1023.59 [M + H +], HPLC (ProtocolEB) retention time = 4.0 minutes (Purity = 96%). 1H NMR (DMSO-d6) 29.88 (d, 1H), 8.48 (d,
[01087] [01087] Preparation of 1,2-dimethyl-L-prolyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3 - {[(2S) - 1 - {[6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexyl] amino} -1-oxo-3-phenylpropan-2-yl] amino} - 1-methoxy -2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (MalC6Am # 151)
[01088] [01088] Step 1. Synthesis of 1,2-dimethyl-L-prolyl-N - [(3R, 4S, 5S) -1 - {(2S) -2 - [(1R, 2R) -3- {[( 2S) -1 - {[6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexyl] amino} -1-oxo-3-phenylpropan-2-yl] amino} - 1-methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-valinamide (MalC6Am # 151). Following general procedure D using # 151 (20 mg, 0.023 mmol, 1.0 eq.), 1- (6-aminohexyl) -1H-pyrrole-2,5-dione (7.0 mg,
[01089] [01089] Preparation of N- [6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] -L-valyl-N- (4 - {(6S, 9R, 10R ) -6-benzyl-10 - [(2S) -1 - {(3R, 4S, 5S) -4 - [(1,2-dimethyl-L-prolyl-L-valyl) (methyl) amino] -3- methoxy-5-methylheptanoyl} pyrrolidin-2-yl] -9-methyl-3,8-dioxo-2,11- dioxa-4,7-diazadodec-1-yl} phenyl) -N ~ 5 ~ -carbamoyl-L -ornitinamide (mcValCitPABC # 246)
[01090] [01090] Step 1. Synthesis of N- [6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] -L-valyl-N- (4 - {(6S, 9R, 10R) -6-benzyl-10 - [(2S) -1 - {(3R, 4S, 5S) -4 - [(1,2-dimethyl-L-prolyl-L-valyl) (methyl) amino] -3-methoxy-5-methylheptanoyl} pyrrolidin-2-yl] -9-methyl-3,8-dioxo-2,11- dioxa-4,7-diazadodec-1-yl} phenyl) -N ~ 5 ~ - carbamoyl-L-ornithinamide (mcValCitPABC # 246). Following general procedure E using # 246 (29.2 mg,
[01091] [01091] In vitro and in vivo HERCEPTIN® studies
[01092] [01092] It should be noted that for the following studies HERCEPTIN® in the absence of conjugated cytotoxic agents does not show any significant in vitro potency or in vivo efficacy at equivalent antibody concentrations.
[01093] [01093] In Vitro Cell Assay Procedure
[01094] [01094] Target expression cells (BT474 (breast cancer), N87 (gastric cancer), HCC1954 (breast cancer), MDA-MB-361-DYT2 (breast cancer)) or non-expression (MDA-MB-468 ) were cultured in 96-well cell culture plates for 24 hours before treatment. Cells were treated with 3-fold serially diluted antibody drug conjugates or free compounds (i.e., no drug-conjugated antibody) in duplicate at 10 concentrations. Cell viability was determined by the CellTiter 96® AQueous One Solution Cell Proliferation MTS Assay (Promega, Madison WI) 96 hours after treatment. Cell relative viability was determined as a percentage of untreated control. IC50 values were calculated using a four-parameter logistic model # 203 with XLfit v4.2 (IDBS, Guildford, Surry, UK). Results are shown in Tables 20, 21A and 21B.
[01095] [01095] MDAMB-361 DYT2 In vivo Tumor Xenograft Model
[01096] [01096] In vivo efficacy studies of drug-antibody conjugates were performed with the Her2 + MDAMB-361 DYT2 cell line. For efficacy studies, 10 million tumor cells in 50% matrigel were implanted subcutaneously in irradiated nude mice 6-8 weeks of age.
[01097] [01097] Six compounds were tested in the three different MDA-MB-361-DYT2 xenograft studies to determine their antitumor activity. The results of a representative study with four of the compounds demonstrate significant tumor regression in mice treated with vehicle during the 50-day observation period (Figure 1). To compare the results of the compounds in the three studies, the antitumor activity was normalized by dividing the volume of the tumor treated with the drug by the volume of the tumor treated with vehicle (T / C). A scheme of six T / C values (Figure 2) shows that each of the six compounds causes complete (or almost complete) regression of the tumor during the observation period, which was up to 107 days, for one of the studies.
[01098] [01098] The test results for H (C) - # D54, H (C) -vcMMAE, H (C) -mcMMAF and H (K) -MCC-DM1 in the MDA-MB-361-DYT2 xenograft studies are shown in Figure 4. Scheme of tumor volume in the treatment group over the control group (T / C) allows comparison between the conjugates (Figure 5C). These results demonstrate that H (C) - # D54 exhibits efficacy equivalent to HERCEPTIN® conjugates with H (C) -vcMMAE, H (C) -mcMMAF and is superior to H (K) -MCC-DM1 in this model.
[01099] [01099] N87 Tumor Xenograft In vivo Model (HERCEPTIN ®)
[01100] [01100] In vivo efficacy studies of antibody-drug conjugates were performed with xenograft models expressing target using N87 cell lines. For the efficacy study, 7.5 million tumor cells in 50% matrigel are implanted subcutaneously in nude mice 6-8 weeks of age, until the tumor sizes reach between 250 and 350 mm3. Dosing is done through bolus injection in the tail vein. Depending on the tumor's response to treatment, animals are injected with 1-10 mg / kg of antibody drug conjugates treated four times every four days. All experimental animals are monitored for changes in body weight per week. The tumor volume is measured twice a week for the first 50 days and once a week thereafter by a Caliper device and calculated with the following formula: tumor volume = (length x width2) / 2. Animals are humanely euthanized before their tumor volumes reach 2500 mm3. The tumor size is seen to decrease after the first week of treatment. Animals can be monitored continuously for tumor regrowth after treatment is stopped.
[01101] [01101] The results of the tests of H (C) - # D54, H (C) -vcMMAE, H (C) -mcMMAF and H (K) -MCC-DM1 in the N87 mouse xenograft selection model are shown in Figures 3 and 5. These results demonstrate that H (C) - # D54 is superior / similar to the H (C) -vcMMAE conjugate and is more potent than H (C) -mcMMAF and H (K) -MCC conjugates -DM1 on this model.
[01102] [01102] Pharmacokinetics and Toxicokinetics
[01103] [01103] Mouse pharmacokinetics and rat toxicokinetics were determined from studies of single dose mouse pharmacokinetics and rat toxicology (see Tables 22 and 23). Mouse pharmacokinetics and rat toxicokinetics were determined in single-dose mouse pharmacokinetics and rat toxicology studies. Mouse pharmacokinetics were determined from samples collected from nude mice that were administered with a single dose of 3 mg / kg. Samples were collected within 336 hours. Rat toxicokinetics were determined in rats (Sprague-Dawley (Crl: CD (SD))), which were administered with a single administration of H (C) -vc- MMAE or H (C) - # D54, in doses of 3 , 10, and 30 mg / kg, or administered with H (C) - mc-DAMM or H (C) -mc-MMAF at 10, 30, and 100 mg / kg. Samples were collected within 336 hours. Circulating concentrations of total antibody and ADC were measured using ELISA assays. The area under the curve (AUC) was calculated for the total antibody and ADC for each ADC. ADC ratios for AUC antibodies were also calculated.
[01104] [01104] Total H (C) - # D54 and ADC antibody exposure was greater than that observed for H (C) -vc-MMAE in mice at 3 mg / kg and at all doses tested in rats. The ADC ratio for AUC Ab to H (C) - # D54 was also greater than that observed for H (C) -vc-MMAE. These results suggest that H (C) - # D54 is more exposed and that ADC and / or ligand load are potentially more stable than H (C) -vc-MMAE.
[01105] [01105] Toxicity
[01106] [01106] Target-independent toxicity of # D54 and ligand load comparators (mcValCitPABC-MMAD and mcValCitPABC-MMAE) conjugated to a non-cross-linked reactive monoclonal antibody (IgG1) were evaluated in a single dose rat toxicity study observation period of two weeks. Doses of antibody-drug conjugates (ADCs) were 0, 3, 10 and 30 mg / kg with n is 5 males / group and the ligand load was similar between the conjugates (3.8, 3.2 and 4, respectively ). These studies included at least daily clinical observations, weekly body weight, clinical pathology (end of life) and necropsy (day 15-17) with microscopic examination of 9 or more tissues and any macroscopic lesions.
[01107] [01107] Mortality with changes in related body weight and signs of morbidity were observed at a dose of 30 mg / kg for all conjugates and at a dose of 10 mg / kg for the MMAD conjugate. There were no clinical observations or changes in body weight in the groups of survivors.
[01108] [01108] The target organs of the conjugates identified by microscopic examination of the surviving dose groups were as follows. The 10 mg / kg conjugate had debris in the epididymis lumen (5/5, minimal to slight), inflammation at the base of the heart (1/5 rats, minimum), and increased corneal mitosis (1/5 rats, Minimum).
[01109] [01109] In summary, the maximum tolerated dose (DMT) of the conjugate and the MMAE conjugate was 10 mg / kg and the DMT of the MMAD conjugate was 3 mg / kg. No adverse effects (NOAEL) of the conjugate were observed to be 3 mg / kg while NOAEL of comparator ligand load conjugates was less than 3 mg / kg.
[01110] [01110] Studies of Anti-IL-13R 2 ADC In Vitro and In Vivo
[01111] [01111] Anti-IL-13R 2 Antibodies and ADCs
[01112] [01112] The humanized antibody hu08 specifically binds to the IL-13R 2 receptor. The amino acid and nucleotide sequences for hu08 are shown in Table 3. Kabat CDRs are underlined.
[01113] [01113] Table 3. Amino acid and nucleotide sequences of humanized hu08 antibodies. SEQ ID DESCRIPTION SEQUENCE
[01114] [01114] Humanized anti-IL-13R 2 antibody hu08 was conjugated to various combinations of ligand charge of the present invention, as provided in Table 4. The antibody drug conjugates were prepared according to the methods of the present invention.
[01115] [01115] Table 4 anti-IL-13R 2 ADCs.
[01116] [01116] In Vitro ICtotoxicity Assay with anti-IL-13R 2 ADCs
[01117] [01117] Cell lines expressing the IL-13R 2 antigen and a negative control cell line, were grown with increased concentrations of anti-IL-13R 2 ADCs comprising the hu08 antibody conjugated to various ligand charges of the present invention. After four days, the viability of each culture was assessed. IC50 values were calculated by non-linear logistic regression and are presented as ng Ab / mL.
[01118] [01118] The data demonstrate that the anti-IL-13R 2 hu08v1.0 / 1.0 antibody conjugated to six different auristatin loads is effective against both tested IL-13R 2 positive cell lines (PC3MM2), having an IC50 ranging from 1.1 at 4.9 ng Ab / mL or 7.3-32.7 pM (Table 5). All ADCs were non-active against IL-13R 2, H460 negative cell line, and non-IL-13R 2 binding control ADCs, huIgG8.84-vc0101 and huIgG8.84-mc3377, were non-active against any of the cell lines tested.
[01119] [01119] In Vivo Subcutaneous Xenograft Models with anti-IL13R 2 ADCs
[01120] [01120] The hu08 humanized antibody specifically binds to the IL-13R 2 receptor. Hu08 ADCs with eleven different ligand load combinations were tested in an xenograft model in vivo. Athymic (nude) female mice were injected subcutaneously with PC3MM2. Mice with staged tumors, approximately 0.1 to 0.3 g (n = 8 to 10 mice / treatment group), were administered intravenously q4d x 4 with normal saline (vehicle), hu08v1.0 / 1.0 ADCs with vc-0101 ligand loads, vc-6780, vc-3906, mc-8261, mc-0131, mc-6121, mc-3377, MalPeg-8261, MalPeg-0131, MalPeg-6121, or MalPeg-3906, and a non-binding Ab (huIgG8. 84) conjugated with vc-0101 or mc-3377, at a dose of 2 or 3 mg Ab / kg. ADCs were dosed based on an Ab content. Tumors were measured at least once a week and their size is calculated as mm3 = 0.5 x (tumor width2) x (tumor length).
[01121] [01121] The in vivo efficacy results listed in Table 6 show a range of antitumor activity with the various tested ADCs. The relative power order is hu08-vc-0101> hu08-vc-6780> hu08-mc-0131> hu08-mc-6121> hu08-mc-3906> hu08-MalPeg-0131> hu08-MalPeg-6121> hu08- MalPeg-3906>> hu08-mc-8261.
[01122] [01122] GT = group finished due to large tumor size
[01123] [01123] ADC Anti-Notch in Vitro and inVivo Studies
[01124] [01124] Anti-Notch Antibody and ADCs
[01125] [01125] Humanized antibodies, hu28 and hu75, and chimeric mouse-human antibodies, ch28 and ch75, specifically bind to the Notch receptor. The amino acid and nucleotide sequences for hu28 and hu75 are provided in Table 7. Kabat CDRs are underlined.
[01126] [01126] Table 7. Humanized anti-Notch antibody amino acid and nucleotide sequences. SEQ DESCRIPTION SEQUENCES ID NO.
[01127] [01127] Humanized anti-Notch antibodies, hu28 and hu75, and chimeric mouse-human anti-Notch antibodies, ch28 and ch75, were conjugated to various combinations of ligand charges of the present invention, as provided in Table 8. The conjugates of antibody drugs were prepared according to the methods of the present invention.
[01128] [01128] In Vitro Cytotoxicity Assays with Anti-Notch ADCs
[01129] [01129] The effects of anti-Notch ADCs were evaluated on 1) cell lines that endogenously express the Notch protein: HCC2429 (lung cancer), OVCAR3 (ovarian cancer) and MDA-MB-468 (breast cancer), 2 ) cell lines built to overexpress Notch protein: MDA-MB- 468 / hNotch and U2OS / hNotch, and 3) a negative control cell line (SW900), using an MTS cell viability indicator (Promega, Madison, WI) . These cell lines were grown with increasing concentrations of anti-Notch ADCs comprising humanized anti-Notch antibodies, and hu28 hu75, and chimeric anti-human rat-Notch antibodies, ch28 and ch75, conjugated to various combinations of ligand charge of the present invention. . As a specificity control for anti-Notch-ADCs, non-target control ADCs (huNeg8.8-ADCs or ch2H6-ADCs) were also tested on the same cell lines. After four days, the viability of each culture was assessed. IC50 values were calculated by logistic non-linear regression and presented as ng Ab / ml. The drug / antibody ratio (DAR) is also provided.
[01130] [01130] Table 9 shows IC50 values (ng Ab / mL) from humanized anti-Notch ADC treatments. Cell lines HCC2429 and MDA-MB-468 / hNotch had two individual replicates. The data demonstrates that humanized anti-Notch ADCs with various ligand loads were active and Notch-induced cell death that expresses and over-expresses cancer cell lines HCC2429, OVCAR3, MDA-MB-468, MDA-MB-468 / hNotch, U2OS / hNotch, but not in the Notch expression missing SW900 from the negative control cell line without.
[01131] [01131] Table 10 shows IC 50 values (ng Ab / ml) of the rat-human chimeric anti-Notch ADC treatments. For experiments with 1 to 4 individual repetitions, mean IC50 were calculated with standard error of the mean (S.E.M.). The data demonstrates that the chimeric anti-Notch mouse-human ADCs with various ligand loads were active and induced cell death in the cancer cell lines that express or overexpress Notch HCC2429, OVCAR3, MDA-MB-468, MDA-MB -468 / hNotch, U2OS / hNotch. Non-target control ADCs also lack power (LP) and therefore IC50 values were not generated as indicated, or were minimally active at the highest doses tested. Anti-Notch ADCs having IC50 values equal to or greater than IC50 values for control ADCs were considered to be underpowered in vitro and indicated as LP.
[01132] [01132] In vivo Human Tumor Xenograft Models with anti-Notch ADCs
[01133] [01133] Humanized anti-Notch antibodies, hu28 and hu75, and chimeric mouse-human anti-Notch antibodies, ch28 and ch75, were conjugated to various combinations of ligand loads and tested in non-lung lung cancer xenograft models small 37622A1 (NSCLC), lung cancer HCC2429, breast cancer MDA-MB-468 and gastric cancer N87. For each model described below, the first dose was given on Day 0. Tumors were measured at least once a week and their volume was calculated with the formula: tumor volume (mm3) = 0.5 x (tumor width2) (tumor length) ). Average tumor volumes (± S.E.M.) for each treatment group were calculated having a maximum of 10 animals and a minimum of 6 animals to be included.
[01134] [01134] The effects of anti-Notch ADCs were examined in immunodeficient mice on the in vivo growth of human tumor xenografts that were established from freshly dried NSCLC 37622A1 tumor fragments obtained according to appropriate consensus procedures (Asterand). Xenografts derived from patients with NSCLC 37622A1 were subcutaneously passed in vivo as animal-to-animal fragments in female nude (Nu / Nu) mice. When the tumors reached a volume of 150 to 300 mm3, they were phased to ensure uniformity of the entire tumor size between the various treatment groups. The NSCLC 37622A1 model was dosed intravenously four times every four days (Q4dx4) with PBS vehicle, humanized anti-Notch ADCs, huNeg-8.8 control ADCs and cisplatin at the doses provided in Table 11.
[01135] [01135] Cisplatin is a platinum-based anticancer agent used in the treatment of cancer and considered a standard care therapy. Cisplatin cross-links DNA in this way inducing apoptosis and inhibiting cell growth. The data demonstrate that anti-Notch ADCs hu28-vc0101, hu28-vc6780, hu75-vc0101 and hu75-vc6780 inhibited growth of NSCLC37622A1 xenografts. In addition, the data show that anti-Notch ADCs inhibited tumor growth more potentially than huNeg8.8 control ADCs. Furthermore, the data show that anti-Notch ADCs inhibited tumor growth more potentially than cisplatin indicating a potency greater than that of a standard platinum-based chemotherapeutic drug.
[01136] [01136] Similar in vivo experiments were performed with the lung cancer cell line HCC2429 as described above. To generate xenografts, female nude (Nu / Nu) mice were implanted subcutaneously with
[01137] [01137] The HCC2429 xenograft model was also dosed intravenously Q4dx4 with PBS vehicle, mouse-human chimeric anti-Notch ADC and huNeg-
[01138] [01138] Similar in vivo experiments were performed with the breast cancer cell line MDA-MB-468 as described above. MDA-MB-468 cells are classified as a baseline type triple-negative breast cancer (TNBC) subtype since they lack expression of the estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 (HER2) ( Lehmann, BD, et al, J Clin Invest. 2011; 121 (7): 2750–2767). To generate xenografts, female SCID Hairless Outbred (SHO) mice were orthopically implanted with 10x106 MDA-MB-468 cells containing 50% Matrigel (BD Biosciences) in the breast fat layer. When the tumors reached a volume of 250 to 450 mm3, the tumors were phased to ensure uniformity of the tumor mass between the various treatment groups. The breast model MDA-MB-468 was dosed intravenously Q4dx4 with vehicle
[01139] [01139] The MDA-MB-468 breast model was also dosed intravenously Q4dx4 with PBS vehicle, mouse-human chimeric anti-Notch ADC and huNeg-
[01140] [01140] Similar in vivo experiments were performed with gastric cancer cell line N87 as described above. To generate xenografts, female nude (Nu / Nu) mice were implanted subcutaneously with 7.5 x106 N87 cells in 50% Matrigel (BD Biosciences). When the tumors reached a volume of 250 to 450 mm3, the tumors were phased to ensure uniformity of the tumor mass between the various treatment groups. The gastric model N87 was dosed intravenously Q4dx4 with PBS vehicle, humanized anti-Notch ADCs,
[01141] [01141] Gastric model N87 was also dosed intravenously Q4dx4 with PBS vehicle, mouse-human chimeric anti-Notch ADC and huNeg-8.8 control ADCs, at a dose of 5mg / kg as provided in Figure 8D. The data demonstrate that mouse-human chimeric anti-Notch ADC with non-cleavable (mc) and cleavable (vc) ligands and various loading combinations inhibited growth of N87 gastric xenografts. In addition, the data show that mouse-human chimeric anti-Notch ADC inhibited tumor growth more potentially than huNeg-
[01142] [01142] The gastric model N87 was also dosed intravenously Q4dx4 with PBS vehicle and chimeric anti-human Notch ADCs ch28-mc0131, ch75-mc0131, ch28-m (H2O) c-0131 and ch75-m (H2O) c -0131 at a dose of 5mg / kg as provided in Figure 8D. The data demonstrate that mouse-human chimeric anti-Notch ADCs with mc0131 and m (H2O) c-0131 ligand loads inhibited growth of N87 gastric xenografts. In addition, the data show that mouse-human chimeric anti-Notch ADCs with c-0131 m (H2O) linker loads were more potent than mouse-human chimeric anti-Notch ADCs with mc0131 linker loads.
[01143] [01143] Table 18A - Selected compounds (cytotoxic peptides with ligands) of the invention
[01144] [01144] Table 19B - Selected conjugates of the invention
Mass Spectrum: Carega or HPLC-SEC retention time and Drug per HPLC mass for ADC-Ligand-Load # heavy chain (HC) antibody ratio (up to 6 difference with theoretical (DAR) mass)
SEC (Protocol O): 7,317
H- (C) _MalPeg3C2- # 41 minutes; HPLC (Protocol P): 4.3
HC mass = 1032
SEC (Protocol O): 7,177
H- (C) _MalPeg6C2- # 42 minutes; HPLC (Protocol P): 3.9
HC mass = 1180
SEC (Protocol O): 7,195
H- (C) _mc- # 44 minutes; HPLC (Protocol P): 4.4 HC mass = 915
SEC (Protocol O): 7,247
H- (C) _MalPeg3C2- # 44 minutes; HPLC (Protocol P): 3.4
HC mass = 1005
SEC (Protocol O): 7,237
H- (C) _MalPeg6C2- # 44 minutes; HPLC (Protocol P): 3.4
HC mass = 1135
SEC (Protocol O): 7,351
H- (C) _mcValCitPABC- # 44 minutes; HPLC (Protocol P): 4.2
HC mass = 1321
SEC (Protocol O): 7.364 H- (C) _Mal-PEG3C2- # 45 minutes; HPLC (Protocol P): 4.3
HC mass = 1017
SEC (Protocol O): 7,419
H- (C) _Mal-PEG6C2- # 45 minutes; HPLC (Protocol P): 3.9
HC mass = 1154
SEC (Protocol O): 7,159
H- (C) _mcValCitPABC- # 45 minutes; HPLC (Protocol P): 4.1
HC mass = 1343
SEC (Protocol O): 7,192
H- (C) _mc- # 54 minutes; HPLC (Protocol P): 4.5
HC mass = 899
SEC (Protocol O): 7,350
H- (C) _Mal-PEG6C2- # 69 minutes; HPLC (Protocol P): 3.4
HC mass = 1122
SEC (Protocol O): 7,254
H- (C) _mcValCitPABC- # 69 minutes; HPLC (Protocol P): 4.5
HC mass = 1305
SEC (Protocol O): 7,466
H- (C) _mcValCitPABC- # 70 minutes; HPLC (Protocol P): 3.7
HC mass = 1318
SEC (Protocol O): 7,478
H- (C) _mc- # 79 minutes; HPLC (Protocol P): 4
HC mass = 946
SEC (Protocol O): 7,635
H- (C) _mcValCitPABC- # 79 minutes; HPLC (Protocol P): 3.7
HC mass = 1349
SEC (Protocol O): 7,510
H- (C) _mc- # 115 minutes; HPLC (Protocol P): 3.54
HC mass = 912
H-A114C- (C114) _mc- # 51 - 2.4
H-A114C- (C114) _mc- # 47 - 2.3
H-A114C- (C114) _mc- # 54 - 2.3
H-A114C- (C114) _mcValCitPABC- - 2 # 47
H-A114C- (C114) _mcValCitPABC- - 1,9 # 54
H-A114C- (C114) _mcValCitPABC- - 2 # 26
H-A114C- (C114) _mc- # 26 - 1,9
H-A114C- (C114) _mcValCitPABC- - 2 # 36
SEC (Protocol P): 7,681 H-A114C- (C114) _mcValCitPABC- minutes; HPLC (Protocol O): 1.95 # 42 HC mass = 1378
H-A114C- (C114) _mc- # 42 - 2
SEC (Protocol P): 7,159
H- (C) -mcValCitPABC- # 54 minutes; HPLC (Protocol O): 4.1
HC mass = 1343
H- (C) _mcValCitPABCAmPeg3C2- - 4.6 # 54
H- (C) _mcValCitPABCAmPeg6C2- - 4,5 # 54
H- (C) _mc- # 47 - 4.2
SEC (Protocol P): 7,179 H- (C) _MalPeg3C2- # 54 minutes; HPLC (Protocol O): 3.7
HC mass = 1028
H- (C) _mc- # 54 - 4
H- (C) _mcValCitPABCAmPeg3C2- - 3.7 # 47
H- (C) _MalPeg3C2- # 47 - 4.3
H- (C) _mcValCitPABCAmPeg3C2- - 4.3 # 42
H- (C) _mc- # 41 - 3.1
H- (C) _mcValCitPABCAmPeg3C2- - 3 # 26
H- (C) _mcValCitPABCAmPeg6C2- - 4.2 # 47
SEC (Protocol P): 7,142
H- (C) _MalPeg3C2- # 42 minutes; HPLC (Protocol O): 4.3
HC mass = 1050
H- (C) _mcValCitPABCAmPeg6C2- 4.2 # 26
H- (C) _mcValCitPABCAmPeg6C2- - 4.1 # 42
SEC (Protocol P): 7,254
H- (C) _MalPeg6C2- # 54 minutes; HPLC (Protocol O): 4.4
HC mass = 1161
SEC (Protocol P): 7.303
H- (C) _MalPeg6C2- # 47 minutes; HPLC (Protocol O): 4.4
HC mass = 1182
H- (C) _MalPeg6C2- # 26 - 4.1
SEC (Protocol P): 7,177
H- (C) -MalPeg6C2- # 42 minutes; HPLC (Protocol O): 3.9
HC mass = 1180
H- (C) _mc- # 36 - 4.2
H- (C) _mcValCitPABC- # 60 - 3.8
H- (C) _MalPeg3C2- # 26 - 3.8
H- (C) _mcValCitPABCAmPeg3C2- - 3,5 # 36
H-A114C-
(C114) _mcValCitPABCAmPeg3C2- - 1,9
# 36
H-A114C- (C114) _MalPeg6C2- # 54 - 2
H- (C) _MalPeg3C2- # 60 - 4.2
H- (C) _MalPeg6C2- # 60 - 4.1
H- (C) _MalPeg6C2- # 41 - 4
SEC (Protocol P): 7,192
H- (C) -mc- # 69 minutes; HPLC (Protocol O): 4.5
HC mass = 899
H- (C) _MalPeg3C2- # 36 - 5.2
H- (C) _mcValCitPABCAmPeg6C2- - 4.2 # 36
H- (C) _MalPeg6C2- # 36 - 5
H- (C) _mcValCitPABCAmPeg3C2- - 4.4 # 41
SEC (Protocol P): 7.317 H- (C) -MalPeg3C2- # 41 minutes; HPLC (Protocol O): 4.3
HC mass = 1032
H- (C) _mcValCitPABCAmPeg6C2- - 4.1 # 60
H-A114C- (C114) _mc- # 66 - 1.8
H-L398C + L443C- - 3.8 (C398 + C443) _mcValCitPABC- # 54
H-K392C + L443C- - 3.8 (C392 + C443) _mcValCitPABC- # 54
SEC (Protocol P): 8,827 H-L443C- (C443) _mcValCitPABC- minutes; HPLC (Protocol O): 2 # 54 HC mass = 1344
H-L398C + V422C- - 3,4 (C398 + C422) _mcValCitPABC- # 54
SEC (Protocol P): 7,195
H- (C) -mc- # 44 minutes; HPLC (Protocol O): 4.4
HC mass = 915
SEC (Protocol P): 7.364 H- (C) -Mal-PEG3C2- # 45 minutes; HPLC (Protocol O): 4.3
HC mass = 1017
H- (C) _2AcAmPeg6C2- # 66 - 4
SEC (Protocol P): 7,419
H- (C) -Mal-PEG6C2- # 45 minutes; HPLC (Protocol O): 3.9
HC mass = 1154
SEC (Protocol P): 7.478 H- (C) -mc- # 79 minutes; HPLC (Protocol O): 4
HC mass = 946
SEC (Protocol P): 7,247
H- (C) -MalPeg3C2- # 44 minutes; HPLC (Protocol O): 3.4
HC mass = 1005
SEC (Protocol P): 7,466
H- (C) -mcValCitPABC- # 70 minutes; HPLC (Protocol O): 3.7
HC mass = 1318
SEC (Protocol P): 7,237
H- (C) -MalPeg6C2- # 44 minutes; HPLC (Protocol O): 3.4
HC mass = 1135
H-A114C- (C114) _mcValCitPABC- - 2 # 69
SEC (Protocol P): 7,635
H- (C) -mcValCitPABC- # 79 minutes; HPLC (Protocol O): 3.7
HC mass = 1349
H-A114C- (C114) _mcValCitPABC- - 1.84 # 79
SEC (Protocol P): 7,351
H- (C) -mcValCitPABC- # 44 minutes; HPLC (Protocol O): 4.2
HC mass = 1321
H-A114C- (C114) _mcValCitPABC- 1.93 # 88
SEC (Protocol P): 7,254
H- (C) -mcValCitPABC- # 69 minutes; HPLC (Protocol O): 4.5
HC mass = 1305
H- (C) _2AcAmCapValCitPABC- # 66 - 3.3
H-A114C- (C114) _mcValCitPABC- - 1.92 # 45
H-A114C- (C114) _mcValCitPABC- - 2 # 34
H-A114C- (C114) _mc- # 45 - 1.95
H-A114C- (C114) _mc- # 70 - 2
SEC (Protocol P): 7,083
H- (C) _mcValCitPABC- # 112 minutes; HPLC (Protocol O): 4.4
HC mass = 1291
SEC (Protocol P): 7,350
H- (C) -Mal-PEG6C2- # 69 minutes; HPLC (Protocol O): 3.4
HC mass = 1122
H-Q347C- (C347) _mcValCitPABC- - 2 # 69
H-Y373C- (C373) _mcValCitPABC- - 1,6 # 69
H-E388C- (C388) _mcValCitPABC- - 2 # 69
H-N421C- (C421) _mcValCitPABC- - 1.95 # 69
H-L443C- (C443) _mcValCitPABC- - 2 # 69
H-L443C- (C443) _mcValCitPABC- - 2 # 79
H-A114C- (C114) _mcValCitPABC- - 2 # 95
H-A114C- (C114) _mcValCitPABC- - 2 # 98
H-A114C- (C114) _MalPeg3C2- # 69 - 2
H-N297Q- (Q) _AmPeg6C2- # 42 - 3.2
H-N297Q- (Q) _AmPeg6C2- # 54 - 3.04
H-N297Q- (Q) _AmPeg6C2- # 47 - 3.16
H-N297Q- (Q) _AmPeg6C2- # 36 - 3.36
H-N297Q- (Q) _AmPeg6C2- # 26 - 3,4
H-N297Q- (Q) _AmPeg6C2- # 66 - 2.8
SEC (Protocol P): 7.012
H-L443C- (C443) _MalPeg6C2- # 69 minutes; HPLC (Protocol O): 2
HC mass = 1120
H-Q347C- (C347) _MalPeg6C2- # 69 - 1,9
H-E388C- (C388) _MalPeg6C2- # 69 - 1.8
H-N421C- (C421) _MalPeg6C2- # 69 - 1.8
H-E380C- (C380) _MalPeg6C2- # 69 - 1.8
H-L398C + L443C- - 3.9 (C398 + C443) _MalPeg6C2- # 69
H-K392C + L443C- - 3,5 (C392 + C443) _MalPeg6C2- # 69
H-kA111C- (kC111) _MalPeg6C2- - 3.7 # 69
H-kK183C- (kC183) _MalPeg6C2- - 2.1 # 69
H-kK207C- (kC207) _MalPeg6C2- - 2,3 # 69
H-A114C- (C114) _mcValCitPABC- - 2 # 108
H-A114C- (C114) _mcValCitPABC- - 1,9 # 84
H-A114C- (C114) _mcValCitPABC- - 1.8 # 226
H-A114C- (C114) _mc- # 108 - 1.9
H-A114C- (C114) _mcValCitPABC- - 1.8 # 117
H-A114C- (C114) _mcValCitPABC- - 1,9 # 115
H-A114C- (C114) _MalPeg6C2- # 98 - 1,9
IL13Ra2-AB08-v1010-hG1- - 3.9 (C) _mcValCitPABC- # 54
IL13Ra2-AB08-v1010-hG1- - 3,5 (C) _mc- # 69
IL13Ra2-AB08-v1010-hG1- - 3,5 (C) _MalPeg6C2- # 69
IL13Ra2-AB08-v1010-hG1- - 4.4 (C) _mcValCitPABC- # 69
H-A114C- (C114) _MalPeg6C2- - 1.9 0 # 118
H-A114C- (C114) _mcValCitPABC- - 1.8 0 # 118
H-A114C- (C114) _mcValCitPABC- - 1.8 # 80
H-A114C- (C114) _mc- # 117 - 1.9
H-A114C- (C114) _mcValCitPABC- - 1.8 # 232
H-A114C- (C114) _MalPeg6C2- - 1,9 # 230
H-A114C- (C114) _MalPeg6C2- - 1,9 # 117
H-A114C- (C114) _mc- # 115 - 2
H-A114C- (C114) _mv- # 115 - 2
H-A114C- (C114) _mb- # 69 - 2
H-A114C- (C114) _mv- # 69 - 2
H-A114C- (C114) _mc-0 # 118 - 2
SEC (Protocol P): 7,797
H- (C) _mc- # 117 minutes; HPLC (Protocol O): 3.5
HC mass = 937
SEC (Protocol P): 8,005
H- (C) _MalPeg6C2- # 117 minutes; HPLC (Protocol O): 3.56 HC mass = 1163
H- (C) _mc-0 # 118 - 4.1
SEC (Protocol P): NA; HPLC
H- (C) _MalPeg6C2-0 # 118 (Protocol O): HC mass = 3.9
1148
IL13Ra2-AB08-v1010-hG1- - 4 (C) _mc-0 # 118
IL13Ra2-AB08-v1010-hG1- - 4.6 (C) _mc- # 226
IL13Ra2-AB08-v1010-hG1- - 3.3 (C) _mc- # 117
IL13Ra2-AB08-v1010-hG1- - 3.3 (C) _MalPeg6C2- # 117
IL13Ra2-AB08-v1010-hG1- - 2.9 (C) _MalPeg6C2-0 # 118
H-A114C- (C114) _MalPeg6C2- - 1,9 # 226
H-A114C- (C114) _mc- # 172 - 1.9
H-A114C- (C114) _mb-0 # 118 - 1.9
H-A114C- (C114) _me-0 # 118 - 2
H-A114C- (C114) _mcValCitPABC- - 1,9 # 134
H-A114C- (C114) _mc- # 131 - 2
H-A114C- (C114) _MalPeg6C2- - 1,9 # 126
H-A114C- (C114) _MalPeg6C2- - 1.7 # 123
H-A114C- (C114) _mc- # 126 - 2
H-A114C- (C114) _mv-0 # 118 - 2
SEC (Protocol P): 7,501
H- (C) _MalPeg6C2- # 226 minutes; HPLC (Protocol O): 4.5
HC mass = 1150
SEC (Protocol P): 7,418
H- (C) _mc- # 226 minutes; HPLC (Protocol O): 4.5
HC mass = 927
IL13Ra2-AB08-v1010-hG1- - 4.2 (C) _MalPeg6C2- # 226
Notch-28-cG1- (C) _mc-0 # 118 - 4.6
SEC (Protocol P): 7.015 Notch-28-cG1- (C) _mc- # 115 minutes; HPLC (Protocol O): 3.7
HC mass = 911
Notch-28-cG1- (C) _MalPeg6C2- - 4.1 0 # 118
SEC (Protocol P): 7,182
Notch-28-cG1- (C) _me-0 # 118 minutes; HPLC (Protocol O): 3.9
HC mass = 937
Notch-75-cG1- (C) _mc-0 # 118 - 3.3
IL13Ra2-19F9-cG1- - 4.1 (C) _mcValCitPABC- # 54
IL13Ra2-19F9-cG1- - 4.2 (C) _mcValCitPABC- # 112
Notch-28-cG1- (C) _mcValCitPABC- - 4.1 # 112
Notch-28-cG1- (C) _MalPeg6C2- # 69 - 4.3
Notch-75-cG1- (C) _MalPeg6C2- # 69 - 3.8
SEC (Protocol P): 7,010
H- (C) _m (H2O) c-0 # 118 minutes; HPLC (Protocol O): 4.1
HC mass = 942
SEC (Protocol P): 6,964
H- (C) _Mal (H2O) Peg6C2-0 # 118 minutes; HPLC (Protocol O): 4
HC mass = 1166
H- (C) _Mal (H2O) Peg6C2- # 69 - 2.8
H- (C) _m (H2O) c- # 69 - 3.6
H- (C) _me-0 # 118 - 4.4
H- (C) _mv-0 # 118 - 4.4
SEC (Protocol P): 7.032 H- (C) _mb-0 # 118 minutes; HPLC (Protocol O): 4.1
HC mass = 896
H-A114C- (C114) _MalC6- # 54 - 1.9
H-A114C- (C114) _mc- # 231 - 1.7
H-A114C- (C114) _MalC6-0 # 118 - 2
SEC (Protocol P): 6,936
H- (C) _Mal (H2O) Peg6C2- # 115 minutes; HPLC (Protocol O): 4.1
HC mass = 1152
H-A114C- (C114) _mc- # 158 - 2
H-A114C- (C114) _mcValCitPABC- - 1,7 # 231
SEC (Protocol P): 6,928
H- (C) _m (H2O) c- # 115 minutes; HPLC (Protocol O): 3.7
HC mass = 930
Notch-75-cG1- (C) _mc- # 115 - 3.7
Notch-75-cG1- (C) _me-0 # 118 - 3.5
Notch-75-cG1- (C) _MalPeg6C2- - 3.8 0 # 118
H-A114C- (C114) _mc- # 237 - 2
H-A114C- (C114) _mc- # 145 - 2
H-A114C- (C114) _MalPeg6C2- - 2 # 145
H-A114C- (C114) _mc- # 162 - 1.9
H-A114C- (C114) _MalC6Am- # 151 - 1,9
Notch-28-cG1- (C) _m (H2O) c-0 # 118 - 3.7
Notch-75-cG1- (C) _m (H2O) c-0 # 118 - 3 H-
(kK188) _COPeg2C2ValCitPABC- - 2
# 54
SEC (Protocol P): 7,766 IL13Ra2-AB08-v1010-hG1- minutes; HPLC (Protocol O): 3.5 (C) _Mal (H2O) Peg6C2-0 # 118 HC mass = 1164
IL13Ra2-AB08-v1010-hG1- - 3.9 (C) _Mal (H2O) Peg6C2- # 115
SEC (Protocol P): 7,813 IL13Ra2-AB08-v1010-hG1- minutes; HPLC (Protocol O): 4.3 (C) _mc- # 115 HC mass = 911
IL13Ra2-AB08-v1010-hG1- - 3.3 (C) _m (H2O) c-0 # 118
H- (C) _mcValCitPABC-0 # 118 - 4.5
SEC (Protocol P): 7,783 IL13Ra2-AB08-v1010-hG1- minutes; HPLC (Protocol O): 3.8 (C) _m (H2O) c- # 115 HC mass = 930
H-A114C- (C114) _mcValCitPABC- - 1,9 # 154
H-A114C- (C114) _MalC6Am- # 153 - 2
IL13Ra2-AB08-v1010-
Q347C + kK183C-hG1- - 4,3 (C347 + kC183) _mcValCitPABC-
# 54
IL13Ra2-AB08-v1010-Q347C-hG1- - 2,1 (C347) _mcValCitPABC- # 54
IL13Ra2-AB08-v1010-hG1-
(kK188) _COPeg2C2AmPeg2C2- - 2
# 69
IL13Ra2-AB08-v1010-hG1-
(kK188) _COPeg2C2ValCitPABC- - 1,9
# 54
IL13Ra2-AB08-v1010-L443C-hG1- - 2,1 (C443) _mcValCitPABC- # 54
IL13Ra2-AB08-v1010-
K392C + L443C-hG1- - 3.7 (C392 + C443) _mcValCitPABC- # 54
IL13Ra2-AB08-v1010-
L443C + kK183C-hG1- - 4 (C443 + kC183) _mcValCitPABC-
# 54
SEC (Protocol P): 7,232
H- (C) _mcValCitPABC- # 98 minutes; HPLC (Protocol O): 4.2
HC mass = 1371
H-A114C- (C114) _mcValCitPABC- - 1,9 # 246
H-H435A- (C) _mcValCitPABC- # 54 - 4
H-M428L + N434S- - 4.2 (C) _mcValCitPABC- # 70
H-M428L + N434S- - 4 (C) _mcValCitPABC- # 54
H-E388C + N421C- - 3,6 (C388 + C421) _mcValCitPABC- # 54
H-Q347C + K392C- - 3.9 (C347 + C392) _mcValCitPABC- # 54
H-L443C + kK183C-
(C443 + kC183) _mcValCitPABC- - 3.7
# 54
H-Q347C + kK183C- SEC (Protocol P): 8.278
(C347 + kC183) _mcValCitPABC- minutes; HPLC (Protocol O): 3.7
# 54 HC mass = 1339
H-Q347C- (C347) _mcValCitPABC- - 1,9 # 54
H-K392C + L443C- - 4 (C392 + C443) _mc- # 115
H-E388C + N421C- - 3.8 (C388 + C421) _mc- # 115
H-Q347C + K392C- - 4 (C347 + C392) _mc- # 115
H-L443C + kK183C- - 3.8 (C443 + kC183) _mc- # 115
H-Q347C + kK183C- - 3.8 (C347 + kC183) _mc- # 115
H-Q347C- (C347) _mc- # 115 - 2
H-kK183C- - 1,9 (kC183) _mcValCitPABC- # 54
H-E388C- (C388) _mcValCitPABC- - 2 # 54
H-kK183C- (kC183) _mc- # 115 - 1.8
SEC (Protocol P): 7.364 H-E388C- (C388) _mc- # 115 minutes; HPLC (Protocol O): 2
HC mass = 914
H-L443C- (C443) _mc- # 115 - 2
H-N421C- (C421) _mcValCitPABC- - 2 # 54
H-N421C- (C421) _mc- # 115 - 2
H-A114C- (C114) _mcGly- # 201 - 1,9
Table 20 - IC50 values for selected compounds (cytotoxic peptides) of the invention
MDA-MB- BT474 N87GMEAN 361-DYT2 Example # GMEAN IC50 (nM) GMEAN IC50 (nM) IC50 (nM)
# 26 0.368 0.543 1.045
# 30 0.682 6.709 1.853
# 34 0.211 1.95 1.199
# 36 0.499 1.205 1.111
# 41 29.666 33.21 51.784
# 42 0.125 0.327 0.195
# 44 7,119 14.61> 16.401
# 45 0.15 0.385 0.415
# 47 <0.244 <0.256 0.317
# 51 <0.599 3.658 -
# 54 <0.133 <0.221 0.206
# 56 0.316 1.256 0.766
# 60 0.524 1.245 0.957
# 66 0.244 0.463 0.334
# 69 80,191 65,979 40,988
# 70 0.179 0.327 0.225
# 75> 100,000> 100,000> 100,000
# 79 0.079 0.137 0.129
# 80 20,346 28,204 32,846
# 84 0.246 0.426 0.686
# 115 31.493 50.302 19.870
# 117 0.096 0.103 0.118
# 118 100,000 100,000 100,000
# 123 0.125 0.089 0.129
# 126 0.315 0.375 0.454
# 130 0.050 0.076 0.039
# 131 0.072 0.185 0.081
# 134 0.108 0.115 0.134
# 140 - - -
# 141 3,367 3,018 -
# 142 0.269 0.259 -
# 143 - - -
# 144 0.172 0.182 0.174
# 145 0.185 0.167 0.229
# 146 0.435 0.195 0.387
# 147 0.456 0.144 0.421
# 148 0.429 0.219 0.502
# 149 0.417 0.250 0.428
# 151 84,867 61,953 84,599
# 153 98,160 47,274 91,350
# 154 0.193 0.572 0.198
# 155 0.323 0.875 0.318
# 158 0.082 0.115 0.100
# 159 0.070 0.075 0.074
# 162 31,448 21,610 27,824
# 163 100,000 72,703 99,433
# 172 0.057 0.144 0.086
# 173 0.088 0.099 0.067
# 178 0.968 1.262 0.911
# 180 0.159 0.117 0.113
# 182 0.153 0.148 0.122
# 184 2,478 5,098 3,427
# 186 - - -
# 188 0.250 0.283 0.404
# 190 0.134 0.066 0.095
# 192 0.262 0.360 0.408
# 194 0.134 0.212 0.198
# 200 0.048 0.029 0.017
# 201 0.144 0.150 0.121
# 207 0.219 0.626 0.260
# 208 0.418 0.379 0.336
# 209 0.067 0.067 0.058
# 217 - - -
# 219 - - -
# 220 35,163 100,000 100,000
# 221 32,402 87,857 65,401
# 222 0.158 0.352 0.272
# 223 7.589 13.026 10.863
# 224 0.383 1.563 0.998
# 225 3,449 10,524 7.575
# 226 0.118 0.478 0.106
# 227 11,008 18,975 12,899
# 228 0.105 0.090 0.078
# 229 18,372 16,566 10,218
# 230 100,000 89,133 70,236
# 231 3.706 15.127 22.855
# 232 0.071 0.194 0.095
# 233 1,074 8,413 5,042
# 234 0.684 0.756 2.004
# 235 0.852 1.320 1.278
# 236 0.020 0.023 0.010
# 237 0.162 0.217 0.278
# 238 0.139 0.077 0.084
# 239 - - -
# 240 11,710 19,930 23,480
# 241 0.364 0.388 0.494
# 242 32,330 41,329 34,529
# 243 1.252 1.301 1.284
# 244 73,123 100,000 100,000
# 245 11.793 33.037 31.856
# 246 3.159 10.828 5.430
# 247 1,007 2,061 1,334
# 257 - - -
Table 21A - IC50 values for selected conjugates of the invention
BT474 HCC1954 N87
IC50 of IC50 of IC50 of
IC50 Anticor IC50 Anticor IC50 Anticorp ADC-Ligand-Load # (nM) po (nM) po (nM) o
(ng / mL) (ng / mL) (ng / mL)
H- (C) _MalPeg3C2- # 41 0.725 25.592 0.465 16.617 4.02 175.448
> 14.09> 6681.1 H- (C) _MalPeg6C2- # 42 0.502 19.855 0.604 24.783 0 50
121.41 493.07> 841.3> 29495, H- (C) _mc- # 44 3.553 14.464 4 7 60 311
114.84 225.59> 440.8> 26346, H- (C) _MalPeg3C2- # 44 2.603 5.113 7 4 81 532
4873.85 H- (C) _MalPeg6C2- # 44 1.318 58.155 1.466 64.663 98.174 1
H- (C) _mcValCitPABC- 0.188 6.717 0.155 5.329 0.781 28.146 # 44
103.76> 740.0> 27967, H- (C) _Mal-PEG3C2- # 45 2.886 1.513 52.791 2 01 742
111.43 9072.13 H- (C) _Mal-PEG6C2- # 45 1,274 49,903 1,423 54,715 4 1
H- (C) _mcValCitPABC- 0.258 9.997 0.204 7.988 0.417 16.737 # 45
H- (C) _mc- # 54 0.436 19.821 0.992 45.072 2.45 138.026
5147.5 2427.63 H- (C) _Mal-PEG6C2- # 69 1.938 0.356 12.995 5.743 4 9
H- (C) _mcValCitPABC- 0.18 7.17 0.073 2.878 <0.185 <8.946 # 69
H- (C) _mcValCitPABC- 0.133 4.522 0.078 2.61 0.249 8.722 # 70
H- (C) _mc- # 79 0.483 18.097 0.654 24.543 7.576 297.254
H- (C) _mcValCitPABC- 0.152 6.634 0.127 5.682 0.489 21.134 # 79
H- (C) _mc- # 115 0.272 - 0.109 - 0.841 -
H-A114C- (C114) _mc- 41,768 - - 17,297 - # 51
H-A114C- (C114) _mc- 3,269 - - 8,216 - # 47
H-A114C- (C114) _mc- 4,294 - - 7,195 - # 54
H-A114C-
(C114) _mcValCitPABC- 0.493 - 0.31 - 0.696 -
# 47
H-A114C-
(C114) _mcValCitPABC- 0.174 - 0.17 - 0.189 -
# 54
H-A114C-
(C114) _mcValCitPABC- 2,548 - 28,2 - 4,314 -
# 26
H-A114C- (C114) _mc-> 1000,> 980.0> 60,648 - - - # 26 00 26
H-A114C-
(C114) _mcValCitPABC- 2.007 - 26.18 - 13.579 -
# 36
H-A114C-
(C114) _mcValCitPABC- 0.283 0.16 - 0.524 -
# 42
H-A114C- (C114) _mc- 0.81 - 1.54 - 44.164 - # 42
H- (C) -mcValCitPABC- 0.292 - 0.27 - 0.345 - # 54 H-
(C) _mcValCitPABCAmP 15,134 - 14,33 - 41,016 -
eg3C2- # 54 H-
(C) _mcValCitPABCAmP 1,898 - 1,4 - 11,71 -
eg6C2- # 54
H- (C) _mc- # 47 4.429 - 3.52 - 20.007 -
> 41.71 H- (C) _MalPeg3C2- # 54 2.181 - 1.54 - - 1
H- (C) _mc- # 54 3.565 - 6.28 - 48.566 -
H-> 1000,> 543.8 (C) _mcValCitPABCAmP 5,228 - - - 00 52 eg3C2- # 47
H- (C) _MalPeg3C2- # 47 1,467 - 1.29 - 16.856 -
H-> 1000, (C) _mcValCitPABCAmP 1,587 - 4.95 - - 000 eg3C2- # 42
H- (C) _mc- # 41 0.506 - 0.68 - 7.543 -
H-> 1000,> 1000, (C) _mcValCitPABCAmP 11,211 - - - 00 000 eg3C2- # 26 H-
(C) _mcValCitPABCAmP 0.935 - 2.46 - 14.283 -
eg6C2- # 47
H- (C) _MalPeg3C2- # 42 0.517 - 0.51 - 5.479 -
H-> 1000,> 1000, (C) _mcValCitPABCAmP 10,992 - - - 00 000 eg6C2- # 26 H-
(C) _mcValCitPABCAmP 1.819 - 1.97 - 75.643 -
eg6C2- # 42
> 56.92 H- (C) _MalPeg6C2- # 54 2.108 - 1.02 - - 8
H- (C) _MalPeg6C2- # 47 1.637 - 1.42 - 31.762 -
> 817.8 H- (C) _MalPeg6C2- # 26 6.385 - 9.55 - - 59
H- (C) -MalPeg6C2- # 42 0.518 - 0.55 -> 7.993 -
> 1000.0> 1000,> 1000, H- (C) _mc- # 36 - - - 00 00 000
H- (C) _mcValCitPABC- 0.835 - 6.45 - 14.917 - # 60
> 1000, H- (C) _MalPeg3C2- # 26 11.506 - 9.43 - - 000
H-> 1000.0> 1000,> 1000, (C) _mcValCitPABCAmP - - - 00 00 000 eg3C2- # 36
H-A114C-> 1000.0> 1000,> 325.7 (C114) _mcValCitPABCA - - - 00 00 14 mPeg3C2- # 36
H-A114C- 133,42 1,228 - 2,01 - - (C114) _MalPeg6C2- # 54 6
> 1000.0> 1000,> 1000, H- (C) _MalPeg3C2- # 60 - - - 00 00 000
> 1000.0> 1000,> 1000, H- (C) _MalPeg6C2- # 60 - - - 00 00 000
H- (C) _MalPeg6C2- # 41 1.166 - 0.36 - 5.882 -
H- (C) -mc- # 69 0.427 - 0.47 - 3.05 -
> 1000,> 1000, H- (C) _MalPeg3C2- # 36 720,826 - - - 00 000
H-> 1000.0> 1000,> 1000, (C) _mcValCitPABCAmP - - - 00 00 000 eg6C2- # 36
> 1000, H- (C) _MalPeg6C2- # 36 878.903 - 159.1 - - 000 H-
(C) _mcValCitPABCAmP 2,363 - 2.28 - 18.728 -
eg3C2- # 41
H- (C) -MalPeg3C2- # 41 0.725 - 0.54 - 4.004 -
H-> 1000, 392.90 (C) _mcValCitPABCAmP 979,982 - - - 00 5 eg6C2- # 60
H-A114C- (C114) _mc-> 1000,> 1000, 17,235 - - - # 66 00 000
H-L398C + L443C-
(C398 + C443) _mcValCit 0.249 - 0.27 - 0.678 -
PABC- # 54
H-K392C + L443C-
(C392 + C443) _mcValCit <0.195 - 0.42 - <0.254 - PABC- # 54
H-L443C-
(C443) _mcValCitPABC- <0.130 - 0.32 - <0.267 -
# 54
H-L398C + V422C-
(C398 + C422) _mcValCit 0.387 - 0.27 - 0.504 -
PABC- # 54
> 507.2> 878.4 H- (C) -mc- # 44 3.553 - - - 3 89
> 834.7 H- (C) -Mal-PEG3C2- # 45 2.886 - 68.41 - - 17
H- (C) _2AcAmPeg6C2-> 1000,> 1000, 703,419 - - - # 66 00 000
> 268.0 H- (C) -Mal-PEG6C2- # 45 1.274 - 2.74 - - 47
H- (C) -mc- # 79 0.483 - 0.65 - 7.576 -
> 440.8 H- (C) -MalPeg3C2- # 44 2.603 - 5.11 - - 81
H- (C) -mcValCitPABC- 0.188 - 0.09 - <0.179 - # 70
H- (C) -MalPeg6C2- # 44 1.318 - 1.47 - 98.174 -
H-A114C-
(C114) _mcValCitPABC- 0.174 - 0.06 - 0.207 -
# 69
H- (C) -mcValCitPABC- 0.152 - 0.15 - 0.459 - # 79
H-A114C-
(C114) _mcValCitPABC- 0.124 - 0.12 - 0.386 -
# 79
H- (C) -mcValCitPABC- 0.252 - 0.18 - 0.732 - # 44
H-A114C-> 1000, (C114) _mcValCitPABC- 8,127 - - 62,825 - 00 # 88
H- (C) -mcValCitPABC- 0.133 - 0.1 - 0.249 - # 69 H-
(C) _2AcAmCapValCitPA 0.436 - 0.99 - 2.45 -
BC- # 66
H-A114C-
(C114) _mcValCitPABC- 0.217 - 0.2 - 0.496 -
# 45
H-A114C-> 1000, (C114) _mcValCitPABC- 3,724 - - 18,422 - 00 # 34
H-A114C- (C114) _mc-> 1000, 148.85 6.431 - - - # 45 00 2
H-A114C- (C114) _mc- 0.349 - 0.62 - 7.208 - # 70
H- (C) _mcValCitPABC- 0,226 - 0,24 - 0,469 - # 112
H- (C) -Mal-PEG6C2- # 69 0.453 - 0.54 - 1.8 -
H-Q347C-
(C347) _mcValCitPABC- 0.368 - 0.06 - 0.22 -
# 69
H-Y373C-
(C373) _mcValCitPABC- 0.359 - 0.06 - 0.295 -
# 69
H-E388C-
(C388) _mcValCitPABC- 0.427 - 0.06 - 0.314 -
# 69
H-N421C-
(C421) _mcValCitPABC- 0.434 - 0.09 - 0.244 -
# 69
H-L443C-
(C443) _mcValCitPABC- 0.239 - 0.05 - 0.272 -
# 69
H-L443C-
(C443) _mcValCitPABC- 0.3 - 0.15 - 0.412 -
# 79
H-A114C-
(C114) _mcValCitPABC- 0.381 - 0.36 - 0.852 -
# 95
H-A114C-
(C114) _mcValCitPABC- 0.171 - 0.24 - 0.258 -
# 98
H-A114C- 0.221 - 0.58 - 1.589 - (C114) _MalPeg3C2- # 69
H-N297Q- 0.466 - 0.36 - 5.42 - (Q) _AmPeg6C2- # 42
H-N297Q- 0.557 - 0.37 - 6.899 - (Q) _AmPeg6C2- # 54
H-N297Q- 0.346 - 0.43 - 4.337 - (Q) _AmPeg6C2- # 47
H-N297Q-> 1000, 284,26 3,003 - - - (Q) _AmPeg6C2- # 36 00 7
H-N297Q- 0.991 - 1.07 - 35.331 - (Q) _AmPeg6C2- # 26
H-N297Q-> 1000,> 1000, 13,812 - - - (Q) _AmPeg6C2- # 66 00 000
H-L443C- 0.251 - 0.25 - 1.989 - (C443) _MalPeg6C2- # 69
H-Q347C- 0.267 - 0.3 - 0.887 - (C347) _MalPeg6C2- # 69
H-E388C- 0.382 - 0.46 - 3.035 - (C388) _MalPeg6C2- # 69
H-N421C- 0.35 - 0.45 - 1.329 - (C421) _MalPeg6C2- # 69
H-E380C- 0.482 - 0.49 - 5.588 - (C380) _MalPeg6C2- # 69
H-L398C + L443C-
(C398 + C443) _MalPeg6 0.226 - 0.3 - 1.346 -
C2- # 69
H-K392C + L443C-
(C392 + C443) _MalPeg6 0.268 - 0.31 - 1.63 -
C2- # 69
H-kA111C-
(kC111) _MalPeg6C2- 0.297 - 0.34 - 1.635 -
# 69
H-kK183C-
(kC183) _MalPeg6C2- 0.257 - 0.5 - 2.23 -
# 69
H-kK207C-
(kC207) _MalPeg6C2- 0.252 - 0.41 - 1.744 -
# 69
H-A114C-
(C114) _mcValCitPABC- 0.212 - 0.12 - 0.777 -
# 108
H-A114C-
(C114) _mcValCitPABC- 0.627 - 12.2 - 1.733 -
# 84
H-A114C-
(C114) _mcValCitPABC- 0,2 - 0,1 - 0,239 -
# 226
H-A114C- (C114) _mc-> 1000.0> 1000, 113.88 - - - # 108 00 00 9
H-A114C-
(C114) _mcValCitPABC- 0.242 - 0.17 - 0.239 -
# 117
H-A114C-
(C114) _mcValCitPABC- 0.202 - 0.2 - 0.211 -
# 115
H-A114C- 0.576 - 0.47 - 1.46 - (C114) _MalPeg6C2- # 98
H-A114C-
(C114) _MalPeg6C2- 0.257 - 0.17 - 0.550 -
0 # 118
H-A114C-
(C114) _mcValCitPABC- 0.251 - 0.24 - 0.398 -
0 # 118
H-A114C-
(C114) _mcValCitPABC- 0.341 - 0.31 - 0.887 -
# 80
H-A114C- (C114) _mc- 0.197 - 0.14 - 0.455 - # 117
H-A114C-
(C114) _mcValCitPABC- 0.376 - 1.31 - 1.367 -
# 232
H-A114C-
(C114) _MalPeg6C2- 0.504 - 0.85 - 3.179 -
# 230
H-A114C-
(C114) _MalPeg6C2- 0.335 - 0.21 - 0.792 -
# 117
H-A114C- (C114) _mc- 0.243 - 0.23 - 0.45 - # 115
H-A114C- (C114) _mv- 0.21 - 0.15 - 0.65 - # 115
H-A114C- (C114) _mb- 0.256 - 0.43 - 2.137 - # 69
H-A114C- (C114) _mv- 0.215 - 0.27 - 1.043 - # 69
H-A114C- (C114) _mc- 0.151 - 0.1 - 0.342 - 0 # 118
H- (C) _mc- # 117 0.162 - 0.06 - 0.314 -
H- (C) _MalPeg6C2- # 117 0.283 - 0.07 - 0.515 -
H- (C) _mc-0 # 118 0.18 - <0.10 - 0.303 -
H- (C) _MalPeg6C2- 0.269 - 0.15 - 0.499 - 0 # 118
H-A114C-
(C114) _MalPeg6C2- 0.28 - 0.22 - 0.685 -
# 226
H-A114C- (C114) _mc- 0.296 - 0.41 - 0.694 - # 172
H-A114C- (C114) _mb- 0.318 - 0.33 - 0.709 - 0 # 118
H-A114C- (C114) _me- 0.256 - 0.33 - 0.64 - 0 # 118
H-A114C-
(C114) _mcValCitPABC- 0.301 - 0.34 - 0.501 -
# 134
H-A114C- (C114) _mc- 0.357 - 0.76 - 1.614 - # 131
H-A114C-
(C114) _MalPeg6C2- 0.284 - 0.36 - 1.377 -
# 126
H-A114C-
(C114) _MalPeg6C2- 0.362 - 0.34 - 1.867 -
# 123
H-A114C- (C114) _mc- 0.319 - 0.49 - 3.294 - # 126
H-A114C- (C114) _mv- 0.209 - 0.25 - 0.719 - 0 # 118
H- (C) _MalPeg6C2- # 226 0.575 - 0.22 - 1.126 -
H- (C) _mc- # 226 0.359 - 0.18 - 0.69 -
H- (C) _m (H2O) c-0 # 118 0.26 - 0.11 - 0.448 - H-
(C) _Mal (H2O) Peg6C2- 0.482 - 0.19 - 0.9 -
0 # 118 H-
(C) _Mal (H2O) Peg6C2- 0.832 - 0.51 - 5.769 -
# 69
H- (C) _m (H2O) c- # 69 0.418 - 0.28 - 1.529 -
H- (C) _me-0 # 118 0.186 - 0.11 - 0.218 -
H- (C) _mv-0 # 118 0.201 - 0.14 - 0.265 -
H- (C) _mb-0 # 118 0.222 - 0.13 - 0.267 -
H-A114C- 0.662 - 5.11 - 8.003 - (C114) _MalC6- # 54
H-A114C- (C114) _mc-> 1000.0> 1000, - -> 1000 - # 231 00 00
H-A114C- 0.976 - 113 - 15.407 - (C114) _MalC6-0 # 118 H-
(C) _Mal (H2O) Peg6C2- 1.06 - 0.28 - 3.439 -
# 115
H-A114C- (C114) _mc- 0.247 - 0.35 - 0.739 - # 158
H-A114C-
(C114) _mcValCitPABC- 1.178 - 24.41 - 13.447 -
# 231
H- (C) _m (H2O) c- # 115 0.393 - 0.17 - 0.498 -
H-A114C- (C114) _mc- 0.97 - 0.68 - 27.907 - # 237
H-A114C- (C114) _mc- 643.39 4,681 - 585.59 - - # 145 1
H-A114C-
(C114) _MalPeg6C2- 12,856 - 190,59 - 89,125 -
# 145
H-A114C- (C114) _mc- 0.377 - 0.15 - 1.144 - # 162
H-A114C- 0.42 - 0.1 - 0.694 - (C114) _MalC6Am- # 151 H-
(kK188) _COPeg2C2Val - - -
CitPABC- # 54
H- (C) _mcValCitPABC- 0.227 - 0.14 - 0.182 - 0 # 118
H-A114C-
(C114) _mcValCitPABC- 0,323 - 0,32 - 0,363 -
# 154
H-A114C- 0.377 - 0.27 - 0.34 - (C114) _MalC6Am- # 153
H- (C) _mcValCitPABC- 0.211 - 0.14 - 0.162 - # 98
H-A114C-
(C114) _mcValCitPABC- 0.357 - 0.65 - 3.197 -
# 246
H-H435A- 0.358 - 0.17 - 0.237 - (C) _mcValCitPABC- # 54
H-M428L + N434S- 0.322 - 0.1 - 0.114 - (C) _mcValCitPABC- # 70
H-M428L + N434S- 0.354 - - 0.217 - (C) _mcValCitPABC- # 54
H-E388C + N421C-
(C388 + C421) _mcValCit 1.38 - 0.99 - 0.855 -
PABC- # 54
H-Q347C + K392C-
(C347 + C392) _mcValCit 0.276 - 0.29 - 0.147 -
PABC- # 54
H-L443C + kK183C-
(C443 + kC183) _mcValCit <0.129 - 0.37 - <0.111 - PABC- # 54
H-Q347C + kK183C-
(C347 + kC183) _mcValCit 0.146 - 0.25 - 0.08 -
PABC- # 54
H-Q347C-
(C347) _mcValCitPABC- 0.153 - 0.33 - 0.111 -
# 54
H-K392C + L443C- 0.323 - 0.1 - 0.304 - (C392 + C443) _mc- # 115
H-E388C + N421C- 1.251 - 0.42 - 0.997 - (C388 + C421) _mc- # 115
H-Q347C + K392C- 0.342 - 0.1 - 0.219 - (C347 + C392) _mc- # 115
H-L443C + kK183C- 0.319 - 0.1 - 0.268 - (C443 + kC183) _mc- # 115
H-Q347C + kK183C- 0.347 - 0.1 - 0.403 - (C347 + kC183) _mc- # 115
H-Q347C- (C347) _mc- 0.272 - 0.18 - 0.278 - # 115
H-kK183C-
(kC183) _mcValCitPABC 0.287 - 0.34 - 0.194 -
- # 54
H-E388C-
(C388) _mcValCitPABC- 0.098 - 0.38 - 0.084 -
# 54
H-kK183C- (kC183) _mc- 0.28 - 0.27 - 0.269 - # 115
H-E388C- (C388) _mc- 0.302 - 0.15 - 0.301 - # 115
H-L443C- (C443) _mc- 0,222 - 0,1 - 0,259 - # 115
H-N421C-
(C421) _mcValCitPABC- <0.051 - 0.42 - <0.051 -
# 54
H-N421C- (C421) _mc- 0.312 - 0.23 - 0.306 - # 115
H-A114C- 0.321 - - - (C114) _mcGly- # 201
Table 21B - IC50 values for selected conjugates of the invention
DYT2 MDA-MB-468
IC50 of IC50 of IC50 IC50 ADC-Ligand-Load # Antibody Antibody (nM) (nM) (ng / mL) (ng / mL)
> 17528.58> 35714.28 H- (C) _MalPeg3C2- # 41> 69.685 - 1 6
> 25857.97 H- (C) _MalPeg6C2- # 42 33.396 5455.61> 629.281 1
> 1000.00> 34090.90> 1000.00> 34090.90 H- (C) _mc- # 44 0 9 0 9
> 1000.00> 44117.64> 1000.00> 44117.64 H- (C) _MalPeg3C2- # 44 0 7 0 7
> 1000.00> 44117.64> 1000.00> 44117.64 H- (C) _MalPeg6C2- # 44 0 7 0 7
> 1000.00> 35714.28 H- (C) _mcValCitPABC- # 44 0.203 7.246 0 6
> 1000.00> 34883.72> 1000.00> 34883.72 H- (C) _Mal-PEG3C2- # 45 0 1 0 1
> 1000.00> 38461.53> 1000.00> 38461.53 H- (C) _Mal-PEG6C2- # 45 0 8 0 8
H- (C) _mcValCitPABC- # 45 0,371 14,304 613,294 24435,914
> 1000.00> 45454.54> 1000.00> 45454.54 H- (C) _mc- # 54 0 5 0 5
> 29849.27> 1000.00> 35714.28 H- (C) _Mal-PEG6C2- # 69> 467.163 9 0 6
H- (C) _mcValCitPABC- # 69 0.156 7.54 547.953 21860.354
> 1000.00> 33333.33 H- (C) _mcValCitPABC- # 70 0.098 3.332 0 3
> 1000.00> 37500.00 H- (C) _mc- # 79 978.508 36694.065 0 0
H- (C) _mcValCitPABC- # 79 0.212 9.528 351.392 15383.462
H- (C) _mc- # 115 0.21 - - -
> 1000.00> 1000.00 H-A114C- (C114) _mc- # 51 - - 0 0
> 1000.00> 1000.00 H-A114C- (C114) _mc- # 47 - - 0 0
> 1000.00> 1000.00 H-A114C- (C114) _mc- # 54 - - 0 0
H-A114C- 383,667 - 445,014 - (C114) _mcValCitPABC- # 47
H-A114C- 0.372 - 362.213 - (C114) _mcValCitPABC- # 54
H-A114C-> 1000.00 -> 930.555 - (C114) _mcValCitPABC- # 26 0
> 1000.00> 1000.00 H-A114C- (C114) _mc- # 26 - - 0 0
H-A114C-> 1000.00> 927.422 - - (C114) _mcValCitPABC- # 36 0
H-A114C- 0.436 - 530.596 - (C114) _mcValCitPABC- # 42
H-A114C- (C114) _mc- # 42> 727,245 - 567,735 -
H- (C) -mcValCitPABC- # 54 0.275 - 471.905 -
H-> 1000.00> 1000.00 (C) _mcValCitPABCAmPeg3C2- - - 0 0 # 54
H-> 1000.00> 1000.00 (C) _mcValCitPABCAmPeg6C2- - - 0 0 # 54
> 1000.00> 1000.00 H- (C) _mc- # 47 - - 0 0
> 1651.00> 1651.00 H- (C) _MalPeg3C2- # 54 - - 7 7
> 1000.00> 1000.00 H- (C) _mc- # 54 - - 0 0
H-> 1000.00> 1000.00 (C) _mcValCitPABCAmPeg3C2- - - 0 0 # 47
> 1000.00> 1000.00 H- (C) _MalPeg3C2- # 47 - - 0 0
H-> 1000.00> 1000.00 (C) _mcValCitPABCAmPeg3C2- - - 0 0 # 42
> 1000.00> 1000.00 H- (C) _mc- # 41 - - 0 0
H-> 1000.00> 1000.00 (C) _mcValCitPABCAmPeg3C2- - - 0 0 # 26
H-> 1000.00> 1000.00 (C) _mcValCitPABCAmPeg6C2- - - 0 0 # 47
H- (C) _MalPeg3C2- # 42 9,675 - 358,435 -
H-> 1000.00> 1000.00 (C) _mcValCitPABCAmPeg6C2- - - 0 0 # 26
H-> 1000.00> 1000.00 (C) _mcValCitPABCAmPeg6C2- - - 0 0 # 42
> 1731.54> 1731.54 H- (C) _MalPeg6C2- # 54 - - 4 4
> 1651.00> 1651.00 H- (C) _MalPeg6C2- # 47 - - 7 7
> 1000.00> 1000.00 H- (C) _MalPeg6C2- # 26 - - 0 0
H- (C) -MalPeg6C2- # 42 5.705 -> 642.029 -
> 1000.00> 1000.00 H- (C) _mc- # 36 - - 0 0
H- (C) _mcValCitPABC- # 60> 699,241 -> 544,495 -
> 1000.00> 1000.00 H- (C) _MalPeg3C2- # 26 - - 0 0
H-> 1000.00> 1000.00 (C) _mcValCitPABCAmPeg3C2- - - 0 0 # 36
H-A114C-> 1000.00> 1000.00 (C114) _mcValCitPABCAmPeg3C - - 0 0 2- # 36
H-A114C- (C114) _MalPeg6C2-> 1000.00> 1000.00 - - # 54 0 0
> 1000.00> 1000.00 H- (C) _MalPeg3C2- # 60 - - 0 0
> 1000.00> 1000.00 H- (C) _MalPeg6C2- # 60 - - 0 0
> 1000.00> 1000.00 H- (C) _MalPeg6C2- # 41 - - 0 0
H- (C) -mc- # 69> 71,831 -> 899,249 -
> 1000.00> 1000.00 H- (C) _MalPeg3C2- # 36 - - 0 0
H-> 1000.00> 1000.00 (C) _mcValCitPABCAmPeg6C2- - - 0 0 # 36
> 1000.00> 1000.00 H- (C) _MalPeg6C2- # 36 - - 0 0
H-> 1000.00> 1000.00 (C) _mcValCitPABCAmPeg3C2- - - 0 0 # 41
> 1000.00 H- (C) -MalPeg3C2- # 41> 69.685 - - 0
H-> 1000.00> 1000.00 (C) _mcValCitPABCAmPeg6C2- - - 0 0 # 60
> 1000.00> 1000.00 H-A114C- (C114) _mc- # 66 - - 0 0
H-L398C + L443C-
(C398 + C443) _mcValCitPABC- 0.463 - 801.354 -
# 54
H-K392C + L443C-
(C392 + C443) _mcValCitPABC- <0.171 - 565.01 -
# 54
H-L443C- (C443) _mcValCitPABC- 0,371 - 500,958 - # 54
H-L398C + V422C-
(C398 + C422) _mcValCitPABC- 0.48 - 610.884 -
# 54
> 1000.00> 1000.00 H- (C) -mc- # 44 - - 0 0
> 1000.00> 1000.00 H- (C) -Mal-PEG3C2- # 45 - - 0 0
> 1000.00> 1000.00 H- (C) _2AcAmPeg6C2- # 66 - - 0 0
> 1000.00> 1000.00 H- (C) -Mal-PEG6C2- # 45 - - 0 0
> 1000.00 H- (C) -mc- # 79 - 978.508 - 0
> 1000.00> 1000.00 H- (C) -MalPeg3C2- # 44 - - 0 0
H- (C) -mcValCitPABC- # 70 0.116 - 547.953 -
> 1000.00> 1000.00 H- (C) -MalPeg6C2- # 44 - - 0 0
H-A114C-> 1000.00 0.083 - - (C114) _mcValCitPABC- # 69 0
H- (C) -mcValCitPABC- # 79 0.212 - 351.392 -
H-A114C- 0.199 - 472.593 - (C114) _mcValCitPABC- # 79
> 1000.00 H- (C) -mcValCitPABC- # 44 0.248 - - 0
H-A114C-> 1000.00> 1000.00 - - (C114) _mcValCitPABC- # 88 0 0
> 1000.00 H- (C) -mcValCitPABC- # 69 0.098 - - 0
H- (C) _2AcAmCapValCitPABC-> 1000.00> 1000.00 - - # 66 0 0
H-A114C- 2.37 -> 968.025 - (C114) _mcValCitPABC- # 45
H-A114C-> 1000.00> 1000.00 - - (C114) _mcValCitPABC- # 34 0 0
> 1000.00> 1000.00 H-A114C- (C114) _mc- # 45 - - 0 0
> 1000.00> 1000.00 H-A114C- (C114) _mc- # 70 - - 0 0
H- (C) _mcValCitPABC- # 112 0.185 -> 563.999 -
H- (C) -Mal-PEG6C2- # 69 0.963 - 748.275 -
H-Q347C-> 1000.00 0.094 - - (C347) _mcValCitPABC- # 69 0
H-Y373C-> 1000.00 0.156 - - (C373) _mcValCitPABC- # 69 0
H-E388C-> 1000.00 0.117 - - (C388) _mcValCitPABC- # 69 0
H-N421C-> 1000.00 0.162 - - (C421) _mcValCitPABC- # 69 0
H-L443C- (C443) _mcValCitPABC-> 1000.00 0.1 - - # 69 0
H-L443C- (C443) _mcValCitPABC- 0.303 - 370.53 - # 79
H-A114C-> 1000.00 61.8 - - (C114) _mcValCitPABC- # 95 0
H-A114C- 0.218 - 609.904 - (C114) _mcValCitPABC- # 98
H-A114C- (C114) _MalPeg3C2-> 1000.00> 1000.00 - - # 69 0 0
> 1000.00> 1000.00 H-N297Q- (Q) _AmPeg6C2- # 42 - - 0 0
> 1000.00> 1000.00 H-N297Q- (Q) _AmPeg6C2- # 54 - - 0 0
> 1000.00> 1000.00 H-N297Q- (Q) _AmPeg6C2- # 47 - - 0 0
> 1000.00> 1000.00 H-N297Q- (Q) _AmPeg6C2- # 36 - - 0 0
> 1000.00> 1000.00 H-N297Q- (Q) _AmPeg6C2- # 26 - - 0 0
> 1000.00> 1000.00 H-N297Q- (Q) _AmPeg6C2- # 66 - - 0 0
H-L443C- (C443) _MalPeg6C2-> 1000.00 - 758.15 - # 69 0
H-Q347C- (C347) _MalPeg6C2- 1,752 - 832.08 - # 69
H-E388C- (C388) _MalPeg6C2- 6,883 -> 973,529 - # 69
H-N421C- (C421) _MalPeg6C2- 1,027 - 472,466 - # 69
H-E380C- (C380) _MalPeg6C2-> 65,641 - 873,254 - # 69
H-L398C + L443C- 0.827 - 846.418 - (C398 + C443) _MalPeg6C2- # 69
H-K392C + L443C-> 32,438 - 804,407 - (C392 + C443) _MalPeg6C2- # 69
H-kA111C- (kC111) _MalPeg6C2- 0,423 - 740,791 - # 69
H-kK183C- (kC183) _MalPeg6C2-> 1000.00 - 749,154 - # 69 0
H-kK207C- (kC207) _MalPeg6C2-> 138,618 - 586,857 - # 69
H-A114C-> 1000.00 - 873.831 - (C114) _mcValCitPABC- # 108 0
H-A114C-> 1000.00 976.796 - - (C114) _mcValCitPABC- # 84 0
H-A114C- 0.101 - 385.851 - (C114) _mcValCitPABC- # 226
> 1000.00> 1000.00 H-A114C- (C114) _mc- # 108 - - 0 0
H-A114C- 0.107 - 469.882 - (C114) _mcValCitPABC- # 117
H-A114C- 0.142 - 989.147 - (C114) _mcValCitPABC- # 115
H-A114C- (C114) _MalPeg6C2-> 1000.00 355.331 - - # 98 0
H-A114C- (C114) _MalPeg6C2- 0.126 -> 865,455 - 0 # 118
H-A114C-> 1000.00 0.215 - - (C114) _mcValCitPABC-0 # 118 0
H-A114C-> 1000.00 0.432 - - (C114) _mcValCitPABC- # 80 0
H-A114C- (C114) _mc- # 117 0.107 -> 414.892 -
H-A114C- 38,422 - 959,259 - (C114) _mcValCitPABC- # 232
H-A114C- (C114) _MalPeg6C2-> 1000.00> 1000.00 - - # 230 0 0
H-A114C- (C114) _MalPeg6C2-> 1000.00 0.179 - - # 117 0
H-A114C- (C114) _mc- # 115 0.238 -> 699.755 -
H-A114C- (C114) _mv- # 115 0.322 -> 668.891 -
> 1000.00> 1000.00 H-A114C- (C114) _mb- # 69 - - 0 0
> 1000.00> 1000.00 H-A114C- (C114) _mv- # 69 - - 0 0
H-A114C- (C114) _mc-0 # 118 0.098 - 432.816 -
H- (C) _mc- # 117 <0.093 - 194.684 -
H- (C) _MalPeg6C2- # 117 <0.080 - 361.061 -
H- (C) _mc-0 # 118 <0.073 - 541.542 -
H- (C) _MalPeg6C2-0 # 118 0,074 - 465,455 -
H-A114C- (C114) _MalPeg6C2- 0,513 - 574,794 - # 226
H-A114C- (C114) _mc- # 172 1.48 - 500.864 -
H-A114C- (C114) _mb-0 # 118 0.208 - 506.604 -
H-A114C- (C114) _me-0 # 118 0.236 - 903.571 -
H-A114C- 1,434 - 648,066 - (C114) _mcValCitPABC- # 134
> 1000.00 H-A114C- (C114) _mc- # 131 - 480.901 - 0
H-A114C- (C114) _MalPeg6C2- 54,268 - 656,645 - # 126
H-A114C- (C114) _MalPeg6C2-> 1000.00 - 543.693 - # 123 0
> 1000.00 H-A114C- (C114) _mc- # 126 - 749.49 - 0
H-A114C- (C114) _mv-0 # 118 0.147 - 490.276 -
H- (C) _MalPeg6C2- # 226 0.206 - 582.309 -
H- (C) _mc- # 226 0.219 - 477.622 -
H- (C) _m (H2O) c-0 # 118 0.071 - 306.626 -
H- (C) _Mal (H2O) Peg6C2-0 # 118 <0.059 - 441.766 -
H- (C) _Mal (H2O) Peg6C2- # 69 0.203 - 459.502 -
H- (C) _m (H2O) c- # 69 0.315 - 740.334 -
H- (C) _me-0 # 118 <0.061 - 455.314 -
H- (C) _mv-0 # 118 0.084 - 531.617 -
H- (C) _mb-0 # 118 0.076 - 584.327 -
H-A114C- (C114) _MalC6- # 54 52,056 - 65,721 -
> 1000.00> 1000.00 H-A114C- (C114) _mc- # 231 - - 0 0
> 1000.00> 1000.00 H-A114C- (C114) _MalC6-0 # 118 - - 0 0
H- (C) _Mal (H2O) Peg6C2- # 115 0.095 - 698.101 -
H-A114C- (C114) _mc- # 158 0.164 - 329.554 -
H-A114C-> 1000.00> 1000.00 - - (C114) _mcValCitPABC- # 231 0 0
H- (C) _m (H2O) c- # 115 <0.069 - 534.743 -
> 1000.00 H-A114C- (C114) _mc- # 237 - 646,464 - 0
> 1000.00> 1000.00 H-A114C- (C114) _mc- # 145 - - 0 0
H-A114C- (C114) _MalPeg6C2-> 1000.00> 1000.00 - - # 145 0 0
H-A114C- (C114) _mc- # 162 0,201 - 676,764 -
H-A114C- (C114) _MalC6Am- # 151 0,469 - 75,696 - H-
(kK188) _COPeg2C2ValCitPABC- - - - -
# 54
> 1000.00 H- (C) _mcValCitPABC-0 # 118 0.081 - - 0
H-A114C- 1,708 - 566,056 - (C114) _mcValCitPABC- # 154
H-A114C- (C114) _MalC6Am- # 153 0.114 - 69.259 -
H- (C) _mcValCitPABC- # 98 0.23 - 270.019 -
H-A114C-> 1000.00> 1000.00 - - (C114) _mcValCitPABC- # 246 0 0
H-H435A- (C) _mcValCitPABC- 0.208 - 339.77 - # 54
H-M428L + N434S- 0.069 - 380.393 - (C) _mcValCitPABC- # 70
H-M428L + N434S- 0.178 - - - (C) _mcValCitPABC- # 54
H-E388C + N421C-
(C388 + C421) _mcValCitPABC- 1,033 - 826,243 -
# 54
H-Q347C + K392C-
(C347 + C392) _mcValCitPABC- 0.103 - 390.7 -
# 54
H-L443C + kK183C-
(C443 + kC183) _mcValCitPABC- <0.103 - 395.707 -
# 54
H-Q347C + kK183C-
(C347 + kC183) _mcValCitPABC- <0.051 - 384.028 -
# 54
H-Q347C- 2.89 - 393.412 - (C347) _mcValCitPABC- # 54
H-K392C + L443C- 0.07 - 542.081 - (C392 + C443) _mc- # 115
H-E388C + N421C-> 1000.00 0.227 - - (C388 + C421) _mc- # 115 0
H-Q347C + K392C- 0.068 - 934.867 - (C347 + C392) _mc- # 115 H-L443C + kK183C- 0.071 - 757.604 - (C443 + kC183) _mc- # 115 H-Q347C + kK183C- 0.073 - 741.434 - (C347 + kC183) _mc- # 115 H-Q347C- (C347) _mc- # 115 0.098 - 888.128 - H-kK183C- 1.339 - 160.012 - (kC183) _mcValCitPABC- # 54 H-E388C- 0.658 - 287.88 - (C388) _mcValCitPABC- # 54 H-kK183C- (kC183) _mc- # 115 0.179 - 775.698 - H-E388C- (C388) _mc- # 115 0.124 - 958.96 - H-L443C- (C443) _mc- # 115 0.108 - 451.857 - H-N421C- 0.601 - 263.107 - (C421) _mcValCitPABC- # 54 H-N421C- (C421) _mc- # 115 0.108 - 668.857 - H-A114C- (C114) _mcGly- # 201 0.073 - - - Table 22 - Values pharmacokinetics selected in mice for conjugates of the invention and selected pharmacokinetic values in mice comprising MMAD, MMAE or MMAF. AUCs were calculated at a 0-last hour of 0- 336 h except where noted.
AUC (0-last) (µg * Hours / mL) ADC / Ab ADC Dose ADC Ab Ratio
H (C) - # D54 10 132001 164001 80 30 378002 417001 91 10 14140 20840 68 H (C) - # A69 30 44040 63480 69 100 146000 212000 69 10 13300 15780 84 H (C) -MalPEG6C2- 30 56 180 80280 93
MMAD 100 134400 146800 92 10 7650 14500 53 H (C) -mc-MMAD 30 20700 43800 47 100 58000 121000 48 3 10803 29503 37 H (C) -vc-MMAE 10 39303 106003 37 30 134003 184003 - 10 10700 24500 44 H (C) -mc-MMAF 30 32000 71500 45 100 83600 176000 48 3 3800 5200 73 H (K) -MCC-DM1 10 12800 16200 79 30 39100 49600 79 1 represents a 0-last 0-312 hours 2 represents a 0 0-168 hours -last 3 represents 0-96 hours 0-last
Table 23 - Pharmacokinetic values selected in mice for conjugates of the invention and for conjugates comprising MMAD, MMAE or MMAF. AUCs were calculated at a 0-last 0-336 h except where noted.
AUC (0-last) (µg * Hours / mL) ADC / Ab ADC Dose ADC Ab Ratio H (C) - # D44 3 10701 27201 39 H (C) - # D70 3 2240 4890 46 H (C) - # D69 3 2490 4770 52 H (C) - # A69 3 3594 5722 63 H (C) -MalPEG6C2- 3 2641 5415 49
MMAD H (C) -mc-MMAD 3 3580 4970 72 H (C) -vc-MMAE 3 1600 3290 49 H (C) -mc-MMAF 3 3080 4800 64 1 represents a 0-last of 0-168 hours
Table 24 - Data showing stability of conjugates prepared using closed versus open ring succinimide-based binders
Herceptin ADC GSH Stability Mouse Mouse
(6d) ADC AUC PK
(% load (ug * h / mL) ADC / Ab remaining on day 6)
mc- # 118 ring 65% 2160 55%
closed ring 87% 3490 65%
Open
MalPeg6C2- # 118 ring 82% 2010 70%
closed ring 100% 3000 77%
open mc- # 8261 ring 51% 3590 52%
closed ring 96% 4470 73%
Open
MalPeg6C2- ring 61% 2950 72%
# 8261 closed ring 104% - -
open mc- # 115 ring - 1930 58%
closed ring - 2330 68%
Open
Table 25A - Selected loads and their synthesis methods
Prepared in the same Amount Method of Example way or in mg purification Method (Yield)
preparation
10.5 mg # 220 example # 107 Method M (43%)
15.2 mg # 221 example # 107 Method M (76%)
Procedure # 222 Method J * 14 mg (39%) general L
Procedure 16.6 mg # 223 Method J * general L (42%)
Procedure 18.8 mg # 224 Method J * general L (68%)
Procedure 17.3 mg # 225 Method J * general L (64%)
chromatography 354 mg # 226 example # 146 silica (78%)
Procedure 19.4 mg # 227 Method J * general L (77%)
# 228 example # 131 Method E1 * 30 mg (51%)
# 229 example # 151 Method J * 16 mg (61%)
Procedure # 230 Method J * 69 mg (42%) general L
Procedure # 231 Method J * 4.2 mg (44%) general L *
113 mg # 232 example # 98 Method J * (50%)
chromatography # 233 example # 146 88 mg (82%) silica
Procedure # 234 Method J * 8.5 mg (78%) general L
Procedure # 235 Method J * 27 mg (77%) general L *
# 236 example # 131 achiral 3.7 mg (14%)
chromatography 38.6 mg # 237 example # 145 silica (93%)
chromatography 419 mg # 238 example # 145 silica (81%)
chromatography 315 mg # 239 example # 130 silica (48%)
# 240 example # 142 Method E1 * 6 mg (20%)
# 241 example # 142 Method E1 * 6 mg (20%)
# 242 example # 145 Method J * 8 mg (10%)
# 243 example # 145 Method J * 12 mg (22%)
# 244 example # 145 Method J * 9.6 mg (20%)
Procedure medium # 245 38 mg (55%) general M pressue C18 medium # 246 example # 130 78 mg (80%) press C18
10.5 mg # 247 example # 178 Method M * (57%)
Table 25B - Selected loads and their IUPAC name and characterization data
Mass spectrum: LC-MS or HPLC observed m / z and retention time in Example Name IUPAC 1 minutes: H NMR (400 MHz, DMSO- d6) unless otherwise specified
2-methylalanyl-N - [(3R, 4S, 5S) -
1 - {(2S) -2 - [(1R, 2R) -3-
{[(2R, 4S) -4-carboxy-1-
phenylpentan-2-yl] amino} -1- HPLC (Protocol CB): m / z 746.51 # 220 methoxy-2-methyl-3- [M + H +] (1.57 minutes) oxopropyl] pyrrolidin-1- il} -3-
methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-
valinamide
2-methylalanyl-N - [(3R, 4S, 5S) -
1 - {(2S) -2 - [(1R, 2R) -3-
(bicycle [1.1.1] pent-1-
ylamino) -1-methoxy-2-methyl-3-HPLC (Protocol DB): m / z 622.42 # 221 oxopropyl] pyrrolidin-1-yl} -3- [M + H +] (1.57 minutes)
methoxy-5-methyl-1-
oxoheptan-4-yl] -N-methyl-L-
valinamide
LC-MS (Protocol H): m / z 744.9
[M + H +] (2.19 minutes), 1H NMR (400
2-methylalanyl-N - [(3R, 4S, 5S) - MHz, CDCl3) δ 7.16-7.22 (m), 6.99-7.08 3-methoxy-1 - {(2S) -2 - (m), 6.42-6.51 (m), 6.10-6.17 (m), 4.87- [(1R, 2R) -1-methoxy-3 - {[(1R) - 4.96 (m), 4.65-4.79 (m), 4.27-4.36 (m), 2-methoxy-2-oxo-1- (1- 4.04-4.27 ( m), 3.95-4.02 (m), 3.87-3.93 phenylcyclopropyl) ethyl] amino} - (m), 3.64-3.84 (m), 3.44-3.57 (m), 3.22- # 222 2-methyl-3- 3.42 (m), 3.08-3.17 (m), 2.98-3.07 (m), oxopropyl] pyrrolidin-1 -il} -5- 2.90-2.93 (m), 2.85-2.89 (m), 2.53-2.57 methyl-1-oxoheptan-4-yl] -N- (m ), 2.35-2.51 (m), 2.19-2.27 (m), 2.02-meth 2.16 (m), 1.93-2.00 (m), 1.77 -1.90 (m), il-L-valinamide 1.57-1.70 (m), 1.35-1.52 (m), 1.26-1.33
(m), 1.19-1.25 (m), 1.11-1.16 (m), 1.03-
1.11 (m), 0.83-1.02 (m), 0.79-0.88 (m).
LC-MS (Protocol H): m / z 744.4
[M + H +] (2.17 minutes), 1H NMR (400
MHz, CD3OD) δ 8.19-8.24 (m), 7.87- 2-methylalanyl-N - [(3R, 4S, 5S) - 7.92 (m), 7.20-7.38 ( m), 4.71-5.04 (m), 3-methoxy-1 - {(2S) -2- 4.61-4.71 (m), 4.47-4.52 (m), 4 , 38-4.44 [(1R, 2R) -1-methoxy-3 - {[(1S) - (m), 4.05-4.13 (m), 3.99-4.04 (m) , 3.90-2-methoxy-2-oxo-1- (1- 3.98 (m), 3.64-3.73 (m), 3.52-3.60 (m), phenylcyclopropyl) ethyl ] amino} - 3.46-3.52 (m), 3.37-3.46 (m), 3.35-3.37 # 223 2-methyl-3- (m), 3.29-3 , 35 (m), 3.24-3.28 (m), 3.15-oxopropyl] pyrrolidin-1-yl} -5- 3.19 (m), 3.08-3.14 (m), 3.01-3.06 (m), methyl-1-oxoheptan-4-yl] -N- 2.84-2.87 (m), 2.43-2.63 (m), 1.96- 2.20 meth (m), 1.68-1.95 (m), 1.60-1.66 (m), 1.52-yl-L-valinamide 1.57 (m), 1.33- 1.44 (m), 1.27-1.32 (m),
1.23-1.27 (m), 1.12-1.17 (m), 1.04-1.10
(m), 0.96-1.03 (m), 0.90-0.96 (m), 0.82-
0.90 (m).
LC-MS (Protocol H): m / z 730.8 [M + H +] (2.15 minutes), 1H NMR (400 MHz, CD3OD) δ 7.09-7.18 (m), 6.95- 7.08 (m), 4.88-4.93 (m), 4.75-4.85 (m), 2-methylalanyl-N - [(3R, 4S, 5S) - 4.72-4, 74 (m), 4.62-4.70 (m), 4.50-4.59 1 - {(2S) -2 - [(1R, 2R) -3 - ({(1R) - (m) , 4.09-4.16 (m), 3.96-4.06 (m), 3.82-1 - [(7R) -bicycle [4.2.0] octa- 3.90 (m), 3 , 67-3.76 (m), 3.58-3.67 (m), 1,3,5-trien-7-yl] -2-methoxy-2- 3.58-3.67 (m) , 3.45-3.54 (m), 3.33-3.44 # 224 oxoethyl} amino) -1-methoxy-2- (m), 3.33-3.44 (m), 3.28 -3.33 (m), 3.10- methyl-3-oxopropyl] pyrrolidin- 3.27 (m), 3.00-3.10 (m), 2.93-3.00 (m), 1 -il} -3-methoxy-5-methyl-1-o 2.75-2.78 (m), 2.56-2.65 (m), 2.36-2.45 xoheptan-4-yl] -N-methyl-L- (m), 2.17-2.35 (m), 1.94-2.16 (m), 1.67- valinamide 1.94 (m), 1.48-1 , 67 (m), 1.27-1.33 (m), 1.23-1.27 (m), 1.17-1.26 (m), 1.08-1.17 (m), 0.98-1.07 (m), 0.86-0.98 (m), 0.77- 0.84 (m).
2-methylalanyl-N - [(3R, 4S, 5S) - 1 - {(2S) -2 - [(1R, 2R) -3 - ({(1S) - 1 - [(7S) -bicycle [4.2. 0] octa- 1,3,5-trien-7-yl] -2-methoxy-2- LC-MS (Protocol H): m / z 730.9 # 225 oxoethyl} amino) -1-methoxy-2- [M + H +] (2.19 minutes) methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-o xoheptan-4-yl] -N-methyl-L-valinamide
LC-MS (Protocol Q): m / z 732.4 N, 2-dimethylalanyl-N- [M + H +] (1.24 minutes), 1H NMR δ [(3R, 4S, 5S) -3-methoxy- 1- 8.47-8.53 (m), 8.24-8.29 (m), 7.81-7.91 {(2S) -2 - [(1R, 2R) -1-methoxy- ( m), 7.14-7.27 (m), 4.54-4.75 (m), 4.44-3 - {[(2S) -1-methoxy-1-oxo-3-4 4.54 (m), 3.94-4.02 (m), 3.72-3.78 (m), # 226 phenylpropan-2-yl] amino} -2- 3.61-3.69 (m), 3.28-3.36 (m), 3.14-3.28 methyl-3-oxopropyl] pyrrolidin- (m), 2.99-3.08 (m), 2.81-2.97 (m ), 2.29-1-yl} -5-methyl-1-oxoheptan-4- 2.57 (m), 2.16-2.29 (m), 1.91-2.16 (m), il] -N-methyl-L-valinamide 1.60-1.87 (m), 1.35-1.53 (m), 0.99-1.33 (m), 0.80-0.99 (m), 0.71-0.80 (m).
2-methylalanyl-N - [(3R, 4S, 5S) - 1 - {(2S) -2 - [(1R, 2R) -3 - ({(1S) - 1 - [(7R) -bicycle [4.2. 0] octa- 1,3,5-trien-7-yl] -2-methoxy-2- LC-MS (Protocol Q): m / z 730.4 # 227 oxoethyl} amino) -1-methoxy-2- [M + H +] (1.29 minutes) methyl-3-oxopropyl] pyrrolidin-1-yl} -3-methoxy-5-methyl-1-o xoheptan-4-yl] -N-methyl-L-valinamide
N, N, 2-trimethylalanyl-N-
[(3R, 4S, 5S) -3-methoxy-1-
{(2S) -2 - [(1R, 2R) -1-methoxy-
3 - {[(2S) -1-methoxy-1-oxo-3-HPLC (Protocol A *): m / z 746.5 [M + H +] # 228 phenylpropan-2-yl] amino} -2- ( 7.103 minutes) methyl-3-oxopropyl] pyrrolidin-
1-yl} -5-methyl-1-oxoheptan-4-
il] -N-methyl
-L-valinamide
N, N, 2-trimethylalanyl-N-
[(3R, 4S, 5S) -1 - {(2S) -2-
[(1R, 2R) -3 - {[(1S) -1-carboxy-
2-phenylethyl] amino} -1-methoxy-LC-MS (Protocol Q1): m / z 732.3 # 229 2-methyl-3- [M + H +] (0.70 minutes) oxopropyl] pyrrolidin-1- il} -3-
methoxy-5-methyl-1-
oxoheptan-4-yl] -N-methyl-L-
valinamide
2-methylalanyl-N - [(3R, 4S, 5S) -
1 - {(2S) -2 - [(1R, 2R) -3 - {[(R) -
carboxy (1-
phenylcyclopropyl) methyl] amino} - HPLC (Protocol G): m / z 730.4 [M + H +] # 230 1-methoxy-2-methyl-3- (1.25 minutes) oxopropyl] pyrrolidin-1-yl} -3-
methoxy-5-methyl-1-
oxoheptan-4-yl] -N-methyl-L-
valinamide
LC-MS (Protocol Q1): m / z 1020.6
[M + H +] (0.83 minutes), 1H NMR (400
MHz, CD3OD) δ 8.19-8.23 (m), 7.99-difluoro {2-methylalanyl-N- 8.07 (m), 7.93-7.98 (m), 7.41- 7.45 (m), [(3R, 4S, 5S) -1 - {(2S) -2- 7.23-7.31 (m), 7.17-7.22 (m), 7.00 -7.04 [(3R, 4R, 7S) -7-benzyl-15- {2- (m), 6.32-6.37 (m), 6.20-6.24 (m), 4, 72- [(3,5-dimethyl-1H-pyrrol-2-yl- 4.93 (m), 4.61-4.69 (m), 4.05-4.17 (m), kappaN) methylidene ] -2H- 3.88-3.93 (m), 3.72-3.81 (m), 3.63-3.70 # 231 pyrrol-5-yl-kappaN} -4-methyl- (m ), 3.56-3.62 (m), 3.48-3.56 (m), 3.25- 5.8.13-trioxo-2-oxa-6,9,12- 3.44 ( m), 3.16-3.25 (m), 3.09-3.14 (m), triazapentadecan 2.98-3.09 (m), 2.81-2.90 (m), 2, 54-2.67 -3-yl] pyrrolidin-1-yl} -3-methoxy- (m), 2.39-2.53 (m), 2.09-2.32 (m), 1.75 - 5-methyl-1-oxoheptan-4-yl] -N- 1.97 (m), 1.60-1.69 (m), 1.52-1.59 (m), methyl-L-valinamidate } boron 1.32-1.44 (m), 1.28-1.32 (m), 1.16-1.21
(m), 0.98-1.09 (m), 0.86-0.98 (m), 0.79-
0.90 (m).
LC-MS (Protocol Q): m / z 769.3
[M + H +] (1.34 minutes), 1H NMR δ
9.04-9.17 (m), 8.88-8.94 (m), 8.70-8.86
(m), 8.62-8.67 (m), 7.79-7.84 (m), 7.76- 2-methyl-D-prolyl-N- 7.79 (m), 7.65 -7.69 (m), 7.61-7.64 (m), [(3R, 4S, 5S) -3-methoxy-1- 7.20-7.31 (m), 7.12-7 , 20 (m), 5.44-5.52 {(2S) -2 - [(1R, 2R) -1-methoxy- (m), 5.34-5.46 (m), 4.70- 4.78 (m), 4.56- 2-methyl-3-oxo-3 - {[(1S) -2- # 232 4.67 (m), 4.47-4.54 (m), 3 , 94-4.04 (m), phenyl-1- (1,3-thiazol-2- 3.76-3.83 (m), 3.52-3.61 (m), 3.36-3 , 52 il) ethyl] amino} propyl] pyrrolidin- (m), 3.28-3.35 (m), 3.10-3.27 (m), 2.93- 1-yl} -5-methyl -1-oxoheptan-4- 3.08 (m), 2.77-2.80 (m), 2.64-2.70 (m), yl] -N-methyl-L-valinamide 2.35- 2.54 (m), 2.09-2.34 (m), 1.96-2.09
(m), 1.54-1.88 (m), 1.38-1.52 (m), 1.18-
1.36 (m), 1.03-1.13 (m), 0.81-1.01 (m),
0.68-0.81 (m).
LC-MS (Protocol Q): m / z 732.2
[M + H +] (1.28 minutes), 1H NMR δ methyl N - {(2R, 3R) -3 - [(2S) -1- 8.48- 8.53 (m), 8.22-8 , 28 (m), 7.80-7.92 {(3R, 4S, 5S) -4 - [{N - [(3- (m), 7.14-7.28 (m), 4.74 -4.79 (m), 4.54-aminooxetan-3-yl) carbonyl] - 4.72 (m), 4.43-4.52 (m), 4.24-4.35 (m), L-valyl} (methyl) amino] -3- 4.07-4.12 (m), 3.94-4.02 (m), 3.72- # 233 methoxy-5- 3.78 (m) , 3.61-3.69 (m), 3.48-3.58 (m), methylheptanoyl} pyrrolidin-2- 3.40-3.48 (m), 3.11-3.35 (m) , 2.98-3.11 il] -3-methoxy-2- (m), 2.75-2.97 (m), 2.64-2.69 (m), 2.30-methylpropanoyl} - L- 2.55 (m), 2.17-2.28 (m), 2.03-2.14 (m), phenylalaninate 1.92-2.02 (m), 1.59-1.87 (m), 1.35-1.54
(m), 1.21-1.33 (m), 1.112-1.20 (m),
1.00-1.09 (m), 0.70-0.98 (m).
LC-MS (Protocol H): m / z 589.9
[M + H + 2] (2.29 minutes), 1H NMR (400
2-methylalanyl-N - {(3R, 4S, 5S) - MHz, CD3OD) δ 8.55-8.61 (m), 8.40-1 - [(2S) -2 - {(3R, 4R, 7S, 12S) - 8.45 (m), 8.34-8.39 (m), 8.23-8.28 (m), 7-benzyl-14- [3-chloro-4- 8.14 -8.19 (m), 7.84-7.95 (m), 7.79-7.84 (propan-2-yloxy) phenyl] -4- (m), 7.71-7.77 ( m), 7.61-7.68 (m), 7.46-methyl-12- [4- (8- 7.52 (m), 7.34-7.40 (m), 7.09- 7.27 (m), methylimidazo [1,2-a] pyridin-2- 7.03-7.09 (m), 4.77-4.90 (m), 4.58-4.77 # 234 yl) benzyl] -5,8,14-trioxo-2,9- (m), 4.43-4.55 (m), 4.17-4.33 (m), 4.07-dioxa-6 , 13-diazatetr 4.16 (m), 4.00-4.07 (m), 3.79-3.85 (m), adecan-3-yl} pyrrolidin-1-yl] -3- 3, 58-3.70 (m), 3.44-3.52 (m), 3.12-3.40 methoxy-5-methyl-1- (m), 2.80-3.12 (m), 2.64-2.71 (m), 2.62- oxoheptan-4-yl} -N-methyl-L- 2.64 (m), 2.38-2.47 (m), 2.00- 2.33 (m), valinamide 1.66-2.00 (m), 1.46-1.63 (m), 1.29-1.44
(m), 1.07-1.16 (m), 0.91-1.07 (m), 0.79-
0.87 (m).
LC-MS (Protocol Q1): m / z 944.3 [M + H +] (0.84 minutes), 1H NMR (400 MHz, CD3OD) δ 8.54-8.59 (m), 8.29- 8.33 (m), 7.87-8.02 (m), 7.80-7.87 (m), 2-methylalanyl-N - [(3R, 4S, 5S) - 7.68-7, 74 (m), 7.62-7.67 (m), 7.20-7.38 1 - {(2S) -2 - [(1R, 2R) -3 - {[(2S) - (m) , 4.98-5.06 (m), 4.84-4.97 (m), 4.66-1 - {[4- (5-fluoro-1,3- 4.79 (m), 4 , 61-4.66 (m), 4.13-4.19 (m), benzothiazol-2-yl) -2- 3.98-4.04 (m), 3.91-3.96 (m ), 3.79-3.85 methylphenyl] amino} -1-oxo-3- (m), 3.64-3.73 (m), 3.38-3.56 (m), 3.34- # 235 phenylpropan-2-yl] amino} -1- 3.38 (m), 3.28-3.34 (m), 3.17-3.27 (m), methoxy-2-methyl-3- 3.12-3.16 (m), 3.03-3.11 (m), 2.99-3.03 oxopropyl] pyrrolidin-1-yl} -3 (m), 2.86-2.87 (m), 2.80-2.82 (m), 2.69- methoxy-5-methyl-1- 2.71 (m), 2.31-2.54 (m), 2.27- 2.31 (m), oxoheptan-4-yl] -N-methyl-L- 2.06-2.27 (m), 1.88-2.00 (m), 1.74-1.88 valinamide (m), 1.64-1.74 (m), 1.59-1.64 (m), 1.50-1.59 (m), 1.27-1.48 (m), 1, 19-1.26 (m), 1.11-1.16 (m), 1.06-1.11 (m), 0.96-1.05 (m), 0.86-0.94 ( m), 0.77-0.83 (m).
1,2-dimethyl-D-prolyl-N- [(3R, 4S, 5S) -3-methoxy-1- {(2S) -2 - [(1R, 2R) -1-methoxy- 3 - {[( 2S) -1-methoxy-1-oxo-3-LC-MS (Protocol Q1): m / z 758.3 # 236 phenylpropan-2-yl] amino} -2- [M + H +] (0.74 minutes ) methyl-3-oxopropyl] pyrrolidin- 1-yl} -5-methyl-1-oxoheptan-4-yl] -N-methyl -L-valinamide
LC-MS (Protocol Q1): m / z 771.2
[M + H +] (0.67 minutes), 1H NMR (400
MHz, CD3OD) δ 7.95-7.96 (m), 7.48- N, 2-dimethylalanyl-N- 7.55 (m), 7.45-7.48 (m), 7.26- 7.31 (m), [(3R, 4S, 5S) -1 - {(2S) -2- 6.94-7.18 (m), 5.45-5.49 (m), 5.19 -5.22 [(1R, 2R) -3 - {[(2S) -3- (1H- (m), 5.11-5.17 (m), 4.97-5.00 (m), 4.78- indol-3-yl) -1-methoxy-1- 4.87 (m), 4.68-4.77 (m), 4.59-4.64 (m), oxopropan-2- yl] amino} -1- 4.27-4.34 (m), 3.99-4.16 (m), 3.84-3.92 # 237 methoxy-2-methyl-3- (m), 3.78-3.82 (m), 3.62-3.78 (m), 3.49-oxopropyl] pyrrolidin-1-yl} -3- 3.59 (m), 3.41-3, 49 (m), 3.20-3.41 (m), methoxy-5-methyl-1- 2.99-3.20 (m), 2.95-2.98 (m), 2.82- 2.86 oxoheptan-4-yl] -N-methyl-L- (m), 2.77-2.79 (m), 2.62-2.68 (m), 2.28-valinamide 2.49 (m), 2.19-2.27 (m), 1.98-2.16 (m),
1.56-1.91 (m), 1.31-1.49 (m), 1.19-1.30
(m), 1.15-1.19 (m), 1.06-1.13 (m), 0.88-
1.03 (m), 0.79-0.87 (m).
LC-MS (Protocol Q1): m / z 758.84 [M + H +] (0.71 minutes), 1H NMR (400 MHz, CD3OD) δ 7.15-7.32 (m), 5.86- N, 2-dimethylalanyl-N- 6.00 (m), 5.28-5.40 (m), 5.17-5.27 (m), [(3R, 4S, 5S) -3-methoxy- 1- 4.97-5.04 (m), 4.69-4.91 (m), 4.57-4.69 {(2S) -2 - [(1R, 2R) -1-methoxy- ( m), 4.05-4.21 (m), 3.91-3.96 (m), 3.79-2-methyl-3-oxo-3 - {[(2S) -1- 3.88 (m), 3.71-3.78 (m), 3.62-3.70 (m), # 238 oxo-3-phenyl-1- (prop-2-en-1- 3.25-3 , 56 (m), 3.15-3.24 (m), 3.08-3.14 iloxy) propan-2- (m), 2.90-3.02 (m), 2.79-2 , 87 (m), 2,42-yl] amino} propyl] pyrrolidin-1- 2.52 (m), 2.20-2.38 (m), 2.12-2.20 (m), il } -5-methyl-1-oxoheptan-4-yl] - 2.03-2.12 (m), 2.00-2.03 (m), 1.71 N-methyl-L-valinamide 2, 1.96 (m), 1.33-1.70 (m), 1.23-1.32 (m), 1.17-1.23 (m), 1.12-1.17 (m) , 1.05 - 1.10 (m), 0.94-1.05 (m), 0.82-0.89 (m).
LC-MS (Protocol Q): m / z 786.6 [M + H +] (1.46 minutes), 1H NMR δ 2-methyl-L-prolyl-N- 8.35-8.42 (m), 8.21- 8.31 (m), 8.14-8.20 [(3R, 4S, 5S) -1 - {(2S) -2- (m), 7.15-7.29 (m) , 4.66-4.76 (m), 4.53- [(1R, 2R) -3 - {[(2S) -1-tert- 4.65 (m), 4.46-4.53 ( m), 4.32-4.42 (m), butoxy-1-oxo-3-phenylpropan- 4.07-4.15 (m), 3.96-4.04 (m), 3.76- 3.82 # 239 2-yl] amino} -1-methoxy-2- (m), 3.41-3.61 (m), 3.30-3.38 (m), 3.16- methyl- 3-oxopropyl] pyrrolidin- 3.30 (m), 3.08-3.15 (m) 2.99-3.08 (m), 1-yl} -3-methoxy-5-methyl-1- 2 , 92-2.96 (m), 2.78-2.90 (m), 2.63-2.78 oxoheptan-4-yl] -N-methyl (m), 2.37-2.58 ( m), 2.18-2.36 (m), 2.03- -L-valinamide 2.13 (m), 1.89-2.01 (m), 1.64-1.88 (m) , 1.35-1.62 (m), 1.31-1.35 (m), 1.17-1.31 (m), 1.03-1.14 (m), 0.70-1 , 01 (m).
LC-MS (Protocol Q1): m / z 798.2 N, 2-dimethylalanyl-N- [M + H +] (0.66 minutes), 1H NMR (400 [(3R, 4S, 5S) -3-methoxy -1- MHz, CD3OD) δ 8.43-8.49 (m), 7.50- {(2S) -2 - [(1R, 2R) -1-methoxy- 7.53 (m), 7, 42-7.48 (m), 7.06-7.20 (m), 2-methyl-3-oxo-3 - ({(2S) -1- 4.21-4.83 (m), 3 , 95-4.13 (m), 3.76-3.88 oxo-3-phenyl-1 - [(1H-1,2,3- (m), 3.53-3.67 (m), 3.16-3.47 (m), 3.08- # 240 triazole-4- 3.15 (m) 3.00-3.16 (m), 2.77-2.90 (m), ilmethyl ) amino] propan-2- 2.70-2.73 (m), 2.62-2.69 (m), 2.45-2.58 yl} amino) propyl] pyrrolidin-1- (m), 2.34-2.41 (m), 2.21-2.29 (m), 2.12-yl} -5-methyl- 2.21 (m), 1.55-2.09 (m) , 1.39-1.54 (m), 1-oxoheptan-4-yl] -N-methyl-L- 1.16-1.36 (m), 1.04-1.14 (m), 0 , 85-0.99 valinamide (m), 0.73-0.80 (m), 0.00-0.02 (m).
LC-MS (Protocol Q1): m / z 755.1 [M + H +] (0.69 minutes), 1H NMR (400 N, 2-dimethylalanyl-N-MHz, CD3OD) δ 8.36-8.67 (m), 7.26- [(3R, 4S, 5S) -3-methoxy-1- 7.50 (m), 7.10-7.26 (m), 5.13-5.17 (m ), {(2S) -2 - [(1R, 2R) -1-methoxy- 4.95-4.99 (m), 4.67-4.84 (m), 4.61-4.66 2 -methyl-3-oxo-3 - {[(2S) -1- (m), 4.50-4.60 (m), 3.77-4.12 (m), 3.69- # 241 oxo -3-phenyl-1- (prop-2-yn-1- 3.75 (m), 3.56-3.66 (m), 3.44-3.54 (m), ylamino) propan-2 - 3.19-3.44 (m) 3.12-3.19 (m), 3.03-3.12 yl] amino} propyl] pyrrolidin-1- (m), 2.74-2.94 (m), 2.37-2.60 (m), 2.14-yl} -5-methyl-1-oxoheptan-4-yl] - 2.36 (m), 1.60-2.13 ( m), 1.47-1.59 (m), N-methyl-L-valinamide 1.19-1.40 (m), 1.11-1.16 (m), 0.88-1.11 (m), 0.75-0.84 (m), 0.02-0.06 (m).
LC-MS (Protocol Q1): m / z 722.95
[M + H +] (0.52 minutes) 1H NMR (400
MHz, CD3OD) δ 8.78-8.86 (m), 8.71- N, 2-dimethylalanyl-N- 8.73 (m), 7.96-8.00 (m), 7.34- 7.40 (m), [(3R, 4S, 5S) -1 - {(2S) -2- 4.74-4.91 (m), 4.67-4.71 (m), 4.55 -4.63 [(1R, 2R) -3 - {[(2S) -3- (1H- (m), 4.13-4.22 (m), 4.04-4.10 (m), 3.97- imidazol-4-yl) -1-methoxy-1- 4.01 (m), 3.84-3.92 (m), 3.66-3.82 (m), oxopropan-2- yl] amino} -1- # 242 3.42-3.64 (m), 3.26-3.42 (m) 3.11-3.21 methoxy-2-methyl-3- (m), 2 , 90-2.92 (m), 2.83-2.84 (m), 2.59-oxopropyl] pyrrolidin-1-yl} -3- 2.64 (m), 2.48-2.56 (m), 2.32-2.41 (m), methoxy-5-methyl-1- 2.09-2.24 (m), 1.99-2.08 (m), 1.68-1 , 95 oxoheptan-4-yl] (m), 1.59-1.66 (m), 1.51-1.58 (m), 1.35-N-methyl-L-valinamide 1.45 ( m), 1.22-1.26 (m), 1.17-1.21 (m),
0.95-1.12 (m), 0.83-0.89 (m).
LC-MS (Protocol Q1): m / z 748.2
[M + H +] (0.52 minutes) 1H NMR (400
MHz, CD3OD), δ 8.91-8.99 (m), 8.42-
8.46 (m), 8.15-8.20 (m), 7.92-8.01 (m), N, 2-dimethylalanyl-N- 7.00-7.10 (m), 6, 64-6.74 (m), 5.22-5.26 [(3R, 4S, 5S) -1 - {(2S) -2- (m), 5.06-5.09 (m), 4 , 79-4.95 (m), 4.65- [(1R, 2R) -3 - {[(2S) -3- (4- 4.79 (m), 4.59-4.65 (m ), 4.12-4.21 (m), hydroxyphenyl) -1-methoxy-1- 4.05-4.12 (m), 3.91-3.99 (m), 3.84-3, 90 oxopropan-2-yl] amino} -1- # 243 (m), 3.67-3.79 (m), 3.60-3.66 (m), 3.39- methoxy-2-methyl- 3- 3.57 (m), 3.34-3.39 (m) 3.29-3.34 (m), oxopropyl] pyrrolidin-1-yl} -3- 3.12-3.27 (m ), 2.98-3.00 (m), 2.78-2.88 methoxy-5-methyl-1- (m), 2.61-2.65 (m), 2.55-2.57 (m), 2.46-oxoheptan-4-yl] - 2.53 (m), 2.10-2.36 (m), 1.68-1.96 (m), N-methyl-L- valinamide 1.61-1.68 (m), 1.55-1.60 (m), 1.35-1.53
(m), 1.19-1.24 (m), 1.14-1.18 (m), 1.08-
1.13 (m), 0.98-1.08 (m), 0.84-0.92 (m).
LC-MS (Protocol Q1): m / z 718.4
[M + H +] (0.66 minutes) 1H NMR (400
MHz, CD3OD), δ 7.87-7.92 (m), 7.71-
7.76 (m), 7.46-7.53 (m), 7.40-7.46 (m), N, 2-dimethylalanyl-N- 7.19-7.33 (m), 4, 81-4.96 (m), 4.68-4.77 [(3R, 4S, 5S) -1 - {(2S) -2- (m), 4.60-4.65 (m), 4 , 47-4.53 (m), 4.01- [(1R, 2R) -3 - {[(1R) -1-carboxy-4.17 (m), 3.94-3.98 (m) , 3.81-3.86 (m), 2-phenylethyl] amino} -1-methoxy- 3.68-3.76 (m), 3.56-3.64 (m), 3.40-3 , 50 # 244 2-methyl-3- (m), 3.36-3.40 (m) 3.26-3.35 (m), 3.23-oxopropyl] pyrrolidin-1-yl} -3- 3.26 (m), 3.16-3.22 (m), 3.12-3.16 (m), methoxy-5-methyl-1- 2.94-3.06 (m), 2, 91-2.93 (m), 2.86-2.88 oxoheptan-4-yl] -N-methyl-L- (m), 2.41-2.66 (m), 2.32-2, 41 (m), 1.97- valinamide 2.23 (m), 1.85-1.97 (m), 1.71-1.85 (m),
1.62-1.68 (m), 1.50-1.61 (m), 1.37-1.46
(m), 0.98-1.14 (m), 0.85-0.92 (m).
LC-MS (Protocol Q): m / z 835.0 [M + Na +] (0.87 minutes) 1H NMR δ 9.58-9.69 (m), 8.84-9.16 (m), 8.69-8.77 1,2-dimethyl-L-prolyl-N- (m), 8.54-8.60 (m), 8.44-8.50 (m), 8.32- [ (3R, 4S, 5S) -3-methoxy-1- 8.42 (m), 8.25-8.30 (m), 7.13-7.31 (m), {(2S) -2- [(1R, 2R) -1-methoxy- 7.00-7.01 (m), 4.97-5.06 (m), 4.88-4.97 2-methyl-3-oxo-3- {[(2S) -1- (m), 4.57-4.75 (m), 4.45-4.57 (m), 3.84- # 245 oxo-3-phenyl-1- (piperazin -1- 4.45 (m), 3.62-3.84 (m), 3.40-3.62 (m), il) propan-2- 3.13-3.33 (m), 2 , 77-3.10 (m), 2.67-2.75 yl] amino} propyl] pyrrolidin-1- (m), 2.47-2.57 (m), 2.38-2.45 ( m), 1.92-yl} -5-methyl-1-oxoheptan-4-yl] 2.35 (m), 1.58-1.88 (m), 1.37-1.55 (m) , -N-methyl-L-valinamide 1.22-1.32 (m), 0.97-1.06 (m), 0.84-0.97 (m), 0.73-0.81 ( m).
LC-MS (Protocol Q): m / z 366.2 [M + H + 2] (0.91 minutes) 1H NMR δ 1,2-dimethyl-L-prolyl-N- 9.56-9.65 ( m), 8.70-8.76 (m), 8.05-8.09 [(3R, 4S, 5S) -1 - {(2S) -2- (m), 7.77-7.92 (m), 7.14-7.30 (m), 4.60- [(1R, 2R) -3 - {[(2S) -1-amino- 4.72 (m), 4.46-4 , 57 (m), 3.61 to 4.39 (m), 3-phenylpropan-2-yl] amino} -1- 3.41 to 3.61 (m), 3.11 to 3.33 (m ), 2.97-3.09 # 246 methoxy-2-methyl-3- (m), 2.79-2.94 (m), 2.63-2.74 (m), 2.38-oxopropyl ] pyrrolidin-1-yl} -3- 2.56 (m), 2.13-2.37 (m), 1.93-2.13 (m), methoxy-5-methyl-1- 1.45 -1.89 (m), 1.21-1.32 (m), 1.09-1.14 oxoheptan-4-yl] -N-methyl-L- (m), 1.03-1.08 (m), 0.84-095 (m), 0.73-valinamide 0.80 (m).
2-methyl-D-prolyl-N-
[(3R, 4S, 5S) -1 - {(2S) -2-
[(1R, 2R) -3 - {[2- (cyclohepta-
2,4,6-trien-1-yl) ethyl] amino} -1-
methoxy-2-methyl-3-HPLC (Protocol DB): m / z 700.51 # 247 oxopropyl] pyrrolidin-1-yl} -3- [M + H +] (2.56 minutes)
methoxy-5-methyl-1-
oxoheptan-4-yl] -N-methyl-L-
valin amide

权利要求:
Claims (31)
[1]
1. Compound CHARACTERIZED because it is of formula I:
I or a pharmaceutically acceptable salt or solvate thereof, where, independently for each occurrence, 1-2 R3A
N R1 W is, O, R3A
N R1 1-2 or O; R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R3A and R3B are any of the following: (i) R3A is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl; and R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; or (ii) R3A and R3B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene; R4A and R4B are any of the following: (i) R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and
R4B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A and R4B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
The R12
N R12 R5 is,,,,,,,,,,,,, C1-C10 heterocyclyl, C3-C8 carbocyclyl and C6-C14 aryl optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from the group that consists of -C1-C8 alkyl, -C1-C8 alkyl-N (R ') 2, -C1-C8 alkyl-C (O) R', -C1-C8 alkyl-C (O) OR '-O- ( C1-C8 alkyl), -C (O) R ', -OC (O) R', -C (O) OR ', -C (O) N (R') 2, -NHC (O) R ', -S (O) 2R ', -S (O) R', -OH, halogen, -N3, -N (R ') 2, -CN, -NHC (= NH) NH2, -NHCONH2, -S (= O) 2R 'and -SR', where each R 'is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl, or two R' can, together with the nitrogen to which they are attached, form a C1 -C10 heterocyclyl;
O
O O R12 R6 N O R12 R6
The or R5 is, R13, or
The R12
N
H R13 optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -C1-C8 alkyl-N (R ') 2, -C1-C8 alkyl-C (O) R ', -C1-C8 alkyl-C (O) OR', -O- (C1-C8 alkyl), -C (O) R ', -OC (O) R', -C (O) OR ', -C (O) N (R ') 2, -NHC (O) R', -S (O) 2R ', -S (O) R', -OH, halogen, -N3, -N (R ') 2, -CN, -NHC (= NH) NH2, -NHCONH2, -S (= O) 2R ', -SR' and arylene-R ', where each R' is independently selected from the group consisting of hydrogen, C1 - C8 alkyl, C1-C8heterocyclyl, C1-C10alkylene-C3-C8heterocyclyl and aryl, or two R 'may, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; R6 is hydrogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -C1-C8 haloalkyl; R12 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl; R13 is C1-C10 heterocyclyl; and X is O.
[2]
2. Compound CHARACTERIZED for being of formula IIa: IIa or a pharmaceutically acceptable salt or solvate thereof, in which, independently for each occurrence,
1-2 R3A
N R1 W is, O, R3A
N R1 1-2 or O;
Y O
Z Z
Y 1 R is, or; Y is -C2-C20 alkylene-, -C2-C20 heteroalkylene-; -C3-C8 carbocyclo-, -arylene-, -C3-C8heterocyclo-, -Cl-C10alkylene-arylene-, -arylene-Cl-Cl0Clalkylene-, -Cl- Cl0alkylene- (C3-C8 carbocycle) -, - (C3- C8carbocycle) -Cl-C10alkylene-, -Cl-Cl0alkylene- (C3-C8 heterocycle) - or - (C3-C8 heterocycle) -Cl-Cl0alkylene-;
H
N
O R10 N O
Zé,,, O
O
O
H
N
NH2 or -NH2;
G is halogen, -OH, -SH or -S-C1-C6 alkyl; R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R3A and R3B are any of the following: (i) R3A is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl or halogen; or (ii) R3A and R3B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene; R4A and R4B are any of the following: (i) R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R4B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A and R4B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
The R12
N R12 R5 is,,,,,,,,,,,
,, C1-C10 heterocyclyl, C3-C8 carbocyclyl and C6-C14 aryl optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from the group consisting of -C1-C8 alkyl, -C1-C8 alkyl-N ( R ') 2, -C1-C8 alkyl-C (O) R', -C1-C8 alkyl-C (O) OR '-O- (C1-C8 alkyl), -C (O) R', -OC (O) R ', -C (O) OR', -C (O) N (R ') 2, -NHC (O) R', -S (O) 2R ', -S (O) R', -OH, halogen, -N3, -N (R ') 2, -CN, -NHC (= NH) NH2, -NHCONH2, -S (= O) 2R' and -SR ', where each R' is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl, or two R 'can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl;
O
O O R12 R6 N O R12 R6
The or R5 is, R13, or
The R12
N
H R13 optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -C1-C8 alkyl-N (R ') 2, -C1-C8 alkyl-C (O) R ', -C1-C8 alkyl-C (O) OR', -O- (C1-C8 alkyl), -C (O) R ', -OC (O) R', -C (O) OR ', -C (O) N (R ') 2, -NHC (O) R', -S (O) 2R ', -S (O) R', -OH, halogen, -N3, -N (R ') 2, -CN, -NHC (= NH) NH2, -NHCONH2, -S (= O) 2R ', -SR' and arylene-R ', where each R' is independently selected from the group consisting of hydrogen, C1 - C8 alkyl, C1-C8heterocyclyl, C1-C10alkylene-C3-C8heterocyclyl and aryl, or two R 'may, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; R6 is hydrogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -C1-C8 haloalkyl;
R12 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl; R13 is C1-C10 heterocyclyl; and R7 is independently selected for each occurrence in the group consisting of F, Cl, I, Br, NO2, CN and CF3; R10 is hydrogen, -Cl-Cl0alkyl, -C3-C8carbocyclyl, -aryl, -Cl-C10heteroalkyl, -C3- C8heterocycle, -Cl-Cl0alkylene-aryl, -arylene-Cl-Cl0alkyl, -Cl-Cl0alkylylene- (C3- C8carbocycle ), - (C3-C8 carbocycle) -Cl-Cl0alkyl, -Cl-Cl0alkylene- (C3-C8heterocycle), and - (C3-C8 heterocycle) -Cl-Cl0alkyl, where aryl in R10 comprising aryl is optionally substituted by [ R7] h; h is 1, 2, 3, 4 or 5; and X is O.
[3]
3. Compound CHARACTERIZED as being of formula IIIa: IIIa or a pharmaceutically acceptable salt or solvate thereof, in which, independently for each occurrence, 1-2 R3A
N R1 W is, O, R3A
N R1 1-2 or O; R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl;
R3A and R3B are any of the following: (i) R3A is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl; or (ii) R3A and R3B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene; R4A and R4B are any of the following: (i) R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R4B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A and R4B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene; R5 is
R11 R11 O, R11,, R11,
Y 11 N N R R11 R11 H
O O N O O N R11 O, H,,,,, N R11
H
s
N or NH-R11, optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -O- (C1-C8 alkyl), -C (O) R ', - OC (O) R ', -C (O) OR', -C (O) NH2, -C (O) NHR ', -C (O) N (R') 2, -NHC (O) R ', -S (O) 2R ', -S (O) R', -OH, halogen, -N3, -NH2, -NH (R '), -N (R') 2, -CN, -NHC (= NH ) NH2,
-NHCONH2, -S (= O) 2R 'and -SR', where each R 'is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl;
Y
Z 11 R is, or; Y is -C2-C20 alkylene-, -C2-C20 heteroalkylene-, C3-C8 carbocyclo-, -arylene-, - C3-C8 heterocyclo-, -Cl-C10alkylene-arylene-, -arylene-Cl-Cl0Clalkylene-, -Cl -Cl0alkylene- (C3-C8carbocycle) -, - (C3-C8carbocycle) -Cl-C10alkylene-, -Cl-Cl0alkylene- (C3-C8heterocycle) -, or - (C3-C8 heterocycle) -Cl-Cl0alkylene-; Ze or -NH2; G is halogen, -OH, -SH or –S-C1-C6alkyl R7 is independently selected for each occurrence in the group consisting of F, Cl, I, Br, NO2, CN and CF3; h is 1, 2, 3, 4 or 5; and
X is O.
[4]
4. Compound CHARACTERIZED for being of formula IIb: IIb or a pharmaceutically acceptable salt or solvate thereof, in which, independently for each occurrence, 1-2 R3A
N R1 W is, O, R3A
N R1 1-2 or O;
Y O
Z Z
Y R1 is, or; Y is -C2-C20 alkylene-, -C2-C20 heteroalkylene-, -C3-C8 carbocyclo-, -Arylene-, -C3-C8 heterocyclo-, -Cl-C10alkylene-arylene-, -arylene-Cl-Cl0Clalkylene-, - Cl- Cl0alkylene- (C3-C8carbocycle) -, - (C3-C8carbocycle) -Cl-C10alkylene-, -Cl-Cl0alkylene- (C3-C8 heterocycle) -, or - (C3-C8 heterocycle) -Cl-Cl0alkylene-;
Z is,,,
O
H
L N
O
H
N
H
L N O O O, NH2 or -NHL; L is an antibody; R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R3A and R3B are any of the following: (i) R3A is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl; or (ii) R3A and R3B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene; R4A and R4B are any of the following: (i) R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R4B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A and R4B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
The R12
N R12 R5 is,,,,
,,,,,,,,,, C1-C10 heterocyclyl, C3-C8 carbocyclyl and C6-C14 aryl optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from the group consisting of -C1-C8 alkyl, -C1-C8 alkyl-N (R ') 2, -C1-C8 alkyl-C (O) R', -C1-C8 alkyl-C (O) OR '-O- (C1-C8 alkyl), -C (O) R ', -OC (O) R', -C (O) OR ', -C (O) N (R') 2, -NHC (O) R ', -S (O) 2R', -S (O) R ', -OH, halogen, -N3, -N (R') 2, -CN, -NHC (= NH) NH2, -NHCONH2, -S (= O) 2R 'and -SR' , in which each R 'is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl, or two R' may, together with the nitrogen to which they are attached, form a heterocyclyl C1-C10;
O
O O R12 R6 N O R12 R6
The or R5 is, R13, or
The R12
N
H R13 optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -C1-C8 alkyl-N (R ') 2, -C1-C8 alkyl-C (O) R ', -C1-C8 alkyl-C (O) OR', -O- (C1-C8 alkyl), -C (O) R ', -OC (O) R', -C (O) OR ', -C (O) N (R ') 2, -NHC (O) R', -S (O) 2R ', -S (O) R', -OH, halogen,
-N3, -N (R ') 2, -CN, -NHC (= NH) NH2, -NHCONH2, -S (= O) 2R', -SR 'and arylene-R', where each R 'is independently selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8 heterocyclyl, C1-C10 alkylene-C3-C8 heterocyclyl and aryl, or two R 'can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; R6 is hydrogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -C1-C8 haloalkyl; R12 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl; R13 is C1-C10 heterocyclyl; and X is O.
[5]
5. Compound CHARACTERIZED for being of formula IIIb: IIIb or a pharmaceutically acceptable salt or solvate thereof, in which, independently for each occurrence, 1-2 R3A
N R1 W is, O, R3A
N R1 1-2 or O; R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R3A and R3B are any of the following:
(i) R3A is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl; or (ii) R3A and R3B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene; R4A and R4B are any of the following: (i) R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R4B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A and R4B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene; R5 is
R11 R11 O, R11,, R11,
Y 11 N N R R11 R11 H
O O N O O N R 11 O, H,,,,, N R11
H
s
N or NH-R11, optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -O- (C1-C8 alkyl), -C (O) R ', - OC (O) R ', -C (O) OR', -C (O) NH2, -C (O) NHR ', -C (O) N (R') 2, -NHC (O) R ', -S (O) 2R ', -S (O) R', -OH, halogen, -N3, -NH2, -NH (R '), -N (R') 2, -CN, -NHC (= NH ) NH2, -NHCONH2, -S (= O) 2R 'and -SR', where each R 'is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl;
R11 is, or; Y is -C2-C20 alkylene-, -C2-C20 heteroalkylene-, -C3-C8 carbocyclo-, -Arylene-, -C3-C8 heterocyclo-, -Cl-C10alkylene-arylene-, -arylene-Cl-Cl0Clalkylene-, - Cl- Cl0alkylene- (C3-C8carbocycle) -, - (C3-C8carbocycle) -Cl-C10alkylene-, -Cl-Cl0alkylene- (C3-C8 heterocycle) -, or - (C3-C8 heterocycle) -Cl-Cl0alkylene-; Z is,,,
O
H
L N
O
H
N
H
L N O O O, NH2 or -NHL; L is an antibody; X is O.
[6]
6. Compound CHARACTERIZED because it is of formula IIc: R3B 'R3A' H O H L Z 'R1' N N N N D D R5 R2 'O O X
O O 1-20
IIc or a pharmaceutically acceptable salt or solvate thereof, where, independently for each occurrence,
O
O O O
H
N
Y N N
H H
O
O
NH
Y R1 'is or O NH2; Y is -C2-C20 alkylene-, -C2-C20 heteroalkylene-, -C3-C8 carbocyclo-, -Arylene-, -C3-C8 heterocyclo-, -Cl-C10alkylene-arylene-, -arylene-Cl-Cl0Clalkylene-, - Cl- Cl0alkylene- (C3-C8carbocycle) -, - (C3-C8carbocycle) -Cl-C10alkylene-, -Cl-Cl0alkylene- (C3-C8heterocycle) -, or - (C3-C8 heterocycle) -Cl-Cl0alkylene-;
O
H O
N N Z ’is O, O,,
O
H
N
O
H
N
H
N O O O, NH2, or –NH-; L is an antibody; D is –C (R4A ’) (R4B’) - or is absent; R2 'is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, or is absent if present; R3A ’and R3B’ are any of the following:
(i) R3A 'is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R3B 'is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl, or R3B' is C2-C4 alkylene and 5-7 membered ring form as indicated per ; or (ii) R3A 'and R3B', taken together, are C2-C8 alkylene or C1-C8 heteroalkylene; R4A 'and R4B' are any of the following: (i) R4A 'is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R4B 'is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A 'and R4B', taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
The R12
N R12 R5 is,,,,,,,,,,,,, C1-C10 heterocyclyl, C3-C8 carbocyclyl and C6-C14 aryl optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from the group that consists of -C1-C8 alkyl, -C1-C8 alkyl-N (R ') 2, -C1-C8 alkyl-C (O) R', -C1-C8 alkyl-C (O) OR '-O- ( C1-C8 alkyl), -C (O) R ', -OC (O) R', -C (O) OR ', -C (O) N (R') 2, -NHC (O) R ', -S (O) 2R ', -S (O) R', -OH, halogen, -N3, -N (R ') 2, -CN, -NHC (= NH) NH2, -NHCONH2, -S (= O) 2R 'and -SR', where each R 'is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl, or two R' can, together with the nitrogen to which they are attached, form a C1 -C10 heterocyclyl;
O
O O R12 R6 N O R12 R6
The or R5 is, R13, or
The R12
N
H R13 optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -C1-C8 alkyl-N (R ') 2, -C1-C8 alkyl-C (O) R ', -C1-C8 alkyl-C (O) OR', -O- (C1-C8 alkyl), -C (O) R ', -OC (O) R', -C (O) OR ', -C (O) N (R ') 2, -NHC (O) R', -S (O) 2R ', -S (O) R', -OH, halogen, -N3, -N (R ') 2, -CN, -NHC (= NH) NH2, -NHCONH2, -S (= O) 2R ', -SR' and arylene-R ', where each R' is independently selected from the group consisting of hydrogen, C1 - C8 alkyl, C1-C8heterocyclyl, C1-C10alkylene-C3-C8heterocyclyl and aryl, or two R 'may, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; R6 is hydrogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -C1-C8 haloalkyl; R12 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl; R13 is C1-C10 heterocyclyl; and X is O.
[7]
7. Compound CHARACTERIZED because it is of formula IIIc:
H O H
N N N W N R5 'R11'Z' L
O O O X 1-20 IIIc or a pharmaceutically acceptable salt or solvate thereof, where, independently for each occurrence, Wé 1-2 R3A
N R1, O, R3A
N R1 1-2 or O; R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R3A and R3B are any of the following: (i) R3A is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl; or (ii) R3A and R3B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene; R4A and R4B are any of the following: (i) R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and
R4B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or
(ii) R4A and R4B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
R5 is optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -O- (C1-C8 alkyl), -C (O) R ',
-OC (O) R ', -C (O) OR', -C (O) NH2, -C (O) NHR ', -C (O) N (R') 2, -NHC (O) R ' , -S (O) 2R ', -S (O) R', -OH, halogen, -N3, -NH2, -NH (R '), -N (R') 2, -CN, -NHC (= NH) NH2,
-NHCONH2, -S (= O) 2R 'and -SR', where each R 'is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl;
O
O O O
H
N
Y N N
H H
O
O
NH
Y R11 'is either O NH2; Y is -C2-C20 alkylene-, -C2-C20 heteroalkylene-, -C3-C8 carbocyclo-, -Arylene-, -C3-C8 heterocyclo-, -Cl-C10alkylene-arylene-, -arylene-Cl-Cl0Clalkylene-, - Cl- Cl0alkylene- (C3-C8carbocycle) -, - (C3-C8carbocycle) -Cl-C10alkylene-, -Cl-Cl0alkylene- (C3-C8 heterocycle) -, or - (C3-C8 heterocycle) -Cl-Cl0alkylene-;
O
H O
N N Z ’is O, O,,
O
H
N
O
H
N
H
N O O O, NH2, or –NH-; L is an antibody; X is O.
[8]
8. Compound CHARACTERIZED because it is of formula IId: R3B 'R3A' H O H L [linker] N N N N D D R5 R2 'O X
O O O 1-20 IId or a pharmaceutically acceptable salt or solvate thereof, where, independently for each occurrence,
L is an antibody; [linker] is a divalent linker; D is –C (R4A ’) (R4B’) - or is absent; R2 'is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, or is absent if present; R3A 'and R3B' are any of the following: (i) R3A 'is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R3B 'is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl, or R3B' is C2-C4 alkylene and 5-7 membered ring form as indicated per ; or (ii) R3A 'and R3B', taken together, are C2-C8 alkylene or C1-C8 heteroalkylene; R4A 'and R4B' are any of the following: (i) R4A 'is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and R4B 'is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R4A 'and R4B', taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
The R12
N R12 R5 is,,,,,,,,
,,,,,, C1-C10 heterocyclyl, C3-C8 carbocyclyl and C6-C14 aryl optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from the group consisting of -C1-C8 alkyl, -C1-C8 alkyl-N (R ') 2, -C1-C8 alkyl-C (O) R', -C1-C8 alkyl-C (O) OR '-O- (C1-C8 alkyl), -C (O) R ', -OC (O) R', -C (O) OR ', -C (O) N (R') 2, -NHC (O) R ', -S (O) 2R', -S (O ) R ', -OH, halogen, -N3, -N (R') 2, -CN, -NHC (= NH) NH2, -NHCONH2, -S (= O) 2R 'and -SR', where each R 'is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl, or two R' can, together with the nitrogen to which they are attached, form a heterocyclyl C1-C10;
O
O O R12 R6 N O R12 R6
The or R5 is, R13, or
The R12
N
H R13 optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -C1-C8 alkyl-N (R ') 2, -C1-C8 alkyl-C (O) R ', -C1-C8 alkyl-C (O) OR', -O- (C1-C8 alkyl), -C (O) R ', -OC (O) R', -C (O) OR ', -C (O) N (R ') 2, -NHC (O) R', -S (O) 2R ', -S (O) R', -OH, halogen, -N3, -N (R ') 2, -CN, -NHC (= NH) NH2, -NHCONH2, -S (= O) 2R ', -SR' and arylene-R ', where each R' is independently selected from the group consisting of hydrogen, C1 - C8 alkyl, C1-C8 heterocyclyl, C1-C10 alkylene-C3-C8 heterocyclyl and aryl, or two R 'can,
together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl; R6 is hydrogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -C1-C8 haloalkyl; R12 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl; R13 is C1-C10 heterocyclyl; and X is O.
[9]
9. Compound CHARACTERIZED because it is of formula IIId:
H O H
N N N W N R5 '[linker] L
O O O X 1-20 IIId or a pharmaceutically acceptable salt or solvate thereof, where, independently for each occurrence, 1-2 R3A
N R1 W is, O, R3A
N R1 1-2 or O; R1 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R2 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl; R3A and R3B are any of the following: (i) R3A is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and
R3B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl; or
(ii) R3A and R3B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
R4A and R4B are any of the following:
(i) R4A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and
R4B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or
(ii) R4A and R4B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene;
R5 is optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, -O- (C1-C8 alkyl), -C (O) R ',
-OC (O) R ', -C (O) OR', -C (O) NH2, -C (O) NHR ', -C (O) N (R') 2, -NHC (O) R ' , -S (O) 2R ',
-S (O) R ', -OH, halogen, -N3, -NH2, -NH (R'), -N (R ') 2, -CN, -NHC (= NH) NH2,
-NHCONH2, -S (= O) 2R 'and -SR', where each R 'is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl;
[linker] is a divalent linker;
L is an antibody; X is O.
[10]
10. Compound, salt or solvate according to any one of claims 1-9, CHARACTERIZED by W being
[11]
Compound, salt or solvate, according to any one of claims 1-9, CHARACTERIZED by W ser.
[12]
12. Compound, salt or solvate according to any one of claims 1-9, CHARACTERIZED by the fact that R2 is hydrogen or C1-C8 alkyl.
[13]
13. Compound, salt or solvate, according to any one of claims 1-9, CHARACTERIZED by the fact that R3A is C1-C8 alkyl, preferably methyl.
[14]
A compound, salt or solvate, according to any one of claims 1-5, 7 or 9, CHARACTERIZED by the fact that R3A and R3B, taken together, are C2-C8 alkylene or C1-C8 heteroalkylene.
[15]
15. Compound, salt or solvate according to any one of claims 1-2, 4, 6 or 8, CHARACTERIZED by the fact that R5 is,, or
[16]
16. A compound, salt or solvate according to any one of claims 5-9, CHARACTERIZED by the fact that the antibody is selected from trastuzumab, oregovomab, edrecolomab, cetuximab, a humanized monoclonal antibody to the vitronectin receptor (v 3); alemtuzumab; a humanized anti-HLA-DR antibody for the treatment of non-Hodgkin's lymphoma; 131I Lym-1; a murine anti-HLA-Dr10 antibody for the treatment of non-Hodgkin's lymphoma; a humanized anti-CD2 mAb for the treatment of Hodgkin's disease or non-Hodgkin's lymphoma; labetuzumab; bevacizumab; ibritumomab tiuxetan; ofatumumab; panitumumab; rituximab; tositumomab; ipilimumab; gemtuzumab; an anti-IL13 antibody; and an anti-Notch antibody.
[17]
17. Compound or a pharmaceutically acceptable salt or solvate thereof, CHARACTERIZED for being selected from:
N N
O S O
H O S H H
H N N N N N NH N N NH H2N N
N N H2N
O O O O O, H2N O O O O O, O O O O O
Y N,
O
H
O H N N H N H2 N N O H
H N
N N O O N N H2 N N O H2N N
O O
O O O NH O O O O O
O , , ,
O
H N
N N O H2N H N
O O N O
O O N O H H2N O H N
NH O
O O O O N N NH H2N N
O O O O
OH O O O
O , , ,
O
H
O H N N H N H2N N O H
N N H N HN N O O H2N N N
O O N
O O O Y O O
O O O S N
NH
O, N,,
O
O H H N H
AT THE
H N N H N H2N N N NH O S N N N O H2N N
O O O O H O O
O O N N O O O O O
O , , ,
O
The H
H N N
N N
N N NH O H H2N O O O
H N O O O O
N N O O H2N N OH NH
O NH O
O O O O OH O O,,,
O O
H H
N N N N O H N H2N N H N NH H2N N N
O O O O O O N
O O O O O
O O O O O O
NH NH
O O O
OH,,,
O O
H H O
N N N N H
HN N N N N
N N
O O O H O O O N
O O O H O O O
O O OH
NH NH
The NH
O O
O , , ,
O
H
N N O
HN N H O
N N H
O O O HN N N N
O O HN N
H O O O
N O O O O
O N O
H NH O O NH2 NH
O
HN,,,
O O
H O H
N N O H N N
HN N H N HN N
N N N
O O O O HN N H O O O
O H O O O
O O O OH
N N NH
O O,,,
O H H H
H N O O
N N H N H N
N N N N N H2N O O H2N N H2N N
O O O O O O O O O, O O O O O, O O O O O,
O
The H
H N N
O H N N N
H N N N
N O O O
N N O O O O O H2N N O H
O O H OH N
O O O O O N O OH
O , , ,
NH O
S O H
O H O H N N
H N H N N
HN N N N N N O
N HN
N O O O
O O O O O
O O O O O O O,, O
N H O,
NH O NH O
H H NH
N N N H O
N
N N N N
O O O O O O N
O OH O O O
O O OH
AT THE ,
N
O H O O H O, O
N
H O,
O F H O
HN F H HN O
N N N N H H
N N N N N
O O O N N
O O O O H
O O O O O O O
O
N N O H O, O H O,,
O O
H H H H
N N N F H O H
N N N N N N
N N HN N N
H H N
O O O O O O O O O
O O O O O,,,
F H O H N N
N O H O H
HN N N H H N
N N N Y N N Y
N N N
The N N
O O O O H H O
O O O O O O O O O,,,
The N H
F H H N N
HN N N S F H O H O O
N N
N HN N N S
O N N N
O O O HN
O O O O O N
O , ,
O
O
O
O
N H O, NH2 H
O O N O O NH2
HN N
N N N
N HN N HN N
The N N
O O O
O O O
O O O
O O
N
O H O, O
N H O, O
N H O,
H O
F H O H N
N O N N
HN N N F H H N
N HN N N N
N O O O
O O O O O O
O OH, O O O O O
OH,
O
O
N H O,
O
H
N N O
HN N H O
N N H O O H2N N N N O O H2N N
O O O
NH O O O O
OH OH
N OH
O H O HN
O , , ,
H O H O H
O H N H N
H N N N N
N N N N H2N N, H2N N H2N
O O O,
O
O O O
O O O O, O O O O O O O O O O
O
H
N N
HN N
O O O
O H O O H
H N O
N NH H N
N O N N H2N N O H2N N
O O O O
O O O O O O O O,
O O,,
H O
N N O
N N H H
N N O
O O O N N H N
O N N O O O O H2N N
OH O O
N O O O O OH
O H O, O
N H O,,
O O
O H H H N H N N + N
N N N N N H2N N H NH O S O O O B- N O
O
O O O F O H
F N N
O , ,
N
The H
O H O N
H N
N N N N H2 N N H2N N
O O O O
O O O O H O O O O O N Cl
The N
O H, O,
N
The H H
H O O N N
N H O O
N O H H
O O N N N
N N N N O H2N O HN N
O O O O O
N O O O
O
Y N
O H O N
F,, H,
O O N O
H O H
H N O H H
N N N O N N N N
N H N O O H N
HN O O N HN N H N
O O O O
O O O O O NH
O O,,,
O O O
O H O H H
H O H N
N N N H N N N O
HN N N N N O HN N
H HN N O O
O O O O O O O O O
O O O
NH, N, OH, and
O
O H H H
H N N N
N N N O H2N N H
O O NH
O O O.
[18]
18. Compound, or a pharmaceutically acceptable salt or solvate thereof, CHARACTERIZED for being selected from:
and
[19]
19. Load linker or a drug-antibody conjugate CHARACTERIZED to comprise a radical of a compound, as defined in any of claims 17 or 18.
[20]
20. Drug-antibody conjugate CHARACTERIZED to comprise a radical of a compound, as defined in any one of claims 1-3.
[21]
21. Compound or a pharmaceutically acceptable salt or solvate of the same CHARACTERIZED because it is selected from:
O O
H H
O N N N
O H O O N N
N N H
N N O O O O
H H S N
O O
NH O NH2,
O O
The H N
N N
N N O
H O O O
The H
The N
OH,
O O
The H N
O O O N N
N O O N O O
H O O
The H
N OH
O O,
O
The H N
N N
O N O
O O H O O
O H O O
N NH
AT THE
N N H
H O OH
O
NH O NH2,
O
The H N
N N
O N O
O O H O O O
O H O
N NH
AT THE
N N H O
H O
O
NH O NH2,
O O
O
The H H
N N N
O O O N N
H H
N N O O O O
N N
The H H
O
NH O NH2,
O
The H N
N N
O N O
O O H O
O H O O
N N NH
N N O
H OH
H O
O
NH O NH2,
O O O
H
N O O
N N N N O H N
N N
O O O N N
O O O O
O O
NH O NH
O O O O
OH OH , ,
O O
H
N N
The N N
O O
O H O O O
AT THE
N
N N H NH
H O O
O
O
NH OH NH2
O ,
N N
ONLY THE
The H
O O N N NH
O H N NH
N N N
N N
N N O O H O O O O
H O O O
O O,,
O
O O
H H
N N N N
N N
The H
O O O O O
Y N,
N
O O S
The H
O O O N N N NH
H H N
N N
N N O O O O
OH OH
NH O NH2,
O
H
N N H2N O O O HN N
O O O O O O
The N
O
N O H S,
O O
The H
N N
The N N
The H
N O O O
The N
O N O H S,
O O
H
The N N
O O O NH O O O HN N
H O O O O O O
N N O N
N N O
O H H N
O O H S
NH H2N O,
O
The H
N N
O O O HN N
O O O O O O
H H N
N N O O
N N N
O H H O H S
O
NH H2 N O,
The H
O O
H N
N O N N
O O N N
H O O
O O O O Y N,
O
H
N N H2N O O O HN N
O O O O O O
The N
O
N O H S,
O
H
The N N
O O O HN N
O O O O O O
H H N
N N O O
N N N
O H H O H S
O
NH H2N O,
O O
H
The N N
O O O NH O O O HN N
H O O O
N N O O N
N N O O
O H H N
O O H S
NH H2 N O,
O O O
H
O O O N N
N O O N N
H
O O O N
O O
N O H S,
O O
H
The N N
O O O N N
H H
N O N O O N
O O N N O
O H H N
O O H S
NH H2N O,
O
H O
O O N N H
O O O N
O O H N
The N
N
O O O N
The S
O ,
O O
H
The N N
O O O N N
H H
N N O O O NH
N N
O H H O
O
NH O NH2,
O O O
H H
O O O N N N
N O O N N
H O
O O O O
O , , , , , , ,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,,
,
,
,
,
,
,,
,
,
,
,
,
,
,
,,
,
,
,
,,
,
,
,
,,
,
,
,
,
,
,
,
,
,,,,
N
H O O H
H N
N O N N S
The N N
H O O
N O O O O O,
N
O O H
H N
N N S
H O O N N
H H O O
N N O O O
The N N
H H
N O O O
O NH H 2N O,
N
O O H
H N
N N S
O O N N
H H H O O
N N O O O
The N N
H H
N O O O
O NH H2N O,
O
H O H O H
N N N N
The OH
The N N
H O O
N O O O O O,
O
H O H O H
N N N N
The OH
The N N
O O O O O O
N O O e.
[22]
22. Drug-antibody conjugate CHARACTERIZED for comprising a radical of a compound, as defined in claim 21.
[23]
23. The compound according to claim 1, CHARACTERIZED by being of formula or a pharmaceutically acceptable salt or solvate thereof.
[24]
24. A compound according to claim 1, CHARACTERIZED by being of formula or a pharmaceutically acceptable salt or solvate thereof.
[25]
25. A compound according to claim 1, CHARACTERIZED by being of formula or a pharmaceutically acceptable salt or solvate thereof.
[26]
26. The compound according to claim 1, CHARACTERIZED by being of formula or a pharmaceutically acceptable salt or solvate thereof.
[27]
27. A compound according to claim 1, CHARACTERIZED by being of formula or a pharmaceutically acceptable salt or solvate thereof.
[28]
28. A compound according to claim 1, CHARACTERIZED by being of formula or a pharmaceutically acceptable salt or solvate thereof.
[29]
29. A compound according to claim 1, CHARACTERIZED by being of formula or a pharmaceutically acceptable salt or solvate thereof.
[30]
A compound according to claim 1, CHARACTERIZED by being of formula or a pharmaceutically acceptable salt or solvate thereof.
[31]
31. Pharmaceutical composition CHARACTERIZED by comprising a compound, as defined in any one of claims 1-29, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable excipient.

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法律状态:
2020-11-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2021-05-11| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-07-27| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI N? 10196/2001, QUE MODIFICOU A LEI N? 9279/96, A CONCESS?O DA PATENTE EST? CONDICIONADA ? ANU?NCIA PR?VIA DA ANVISA. CONSIDERANDO A APROVA??O DOS TERMOS DO PARECER N? 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL N? 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVID?NCIAS CAB?VEIS. |

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2021-08-31| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2021-11-23| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201161561255P| true| 2011-11-17|2011-11-17|

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US61/561.225|2011-11-17|

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US201261676423P| true| 2012-07-27|2012-07-27|

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US61/676.423|2012-07-27|

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PCT/IB2012/056224|WO2013072813A2|2011-11-17|2012-11-07|Cytotoxic peptides and antibody drug conjugates thereof|

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